Fhm, a novel member of the TNF ligand supergene family

ABSTRACT

The present invention provides a purified polynucleotide encoding a novel polypeptide, designated Fhm, which belongs to the TNF gene superfamily; to purified Fhm polypeptide molecules; to antibodies that bind Fhm; to materials comprising such molecules; and to methods of using such molecules.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.:09/632,287, filed Aug. 3, 2000, now U.S. Pat. No. 6,521,422, whichclaims the benefit of U.S. Provisional Patent Application No.:60/147,294, filed Aug. 4, 1999.

FIELD OF THE INVENTION

The present invention is in the field of recombinant genetics. Inparticular, the present invention relates to a novel receptor ligand,designated Fhm, belonging to the TNF ligand supergene family and nucleicacid molecules encoding the same. The invention also relates to vectors,host cells, anti-Fhm antibodies, and recombinant methods for producingFhm polypeptides. The invention also relates to the use of therecombinant Fhm polypeptide to identify putative bindingpartners/receptors. In addition, provided for are methods and reagentsfor the diagnosis of diseases associated with abnormal Fhm or abnormalexpression of its putative receptor, and methods and pharmaceuticalcompositions for the treatment of diseases associated with abnormal Fhmor abnormal expression of Fhm and/or its receptor. The invention alsodiscloses pharmaceutical compositions for use in the treatment of thesediseases.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression andmanipulation of nucleic acid molecules have greatly accelerated thediscovery of novel therapeutics based upon deciphering the human genome.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates, and coupled with computationalanalyses, allow the assembly of overlapping sequences into entire genomeand the identification of polypeptide-encoding regions. Comparison of apredicted amino acid sequence against a database compilation of knownamino acid sequences can allow one to determine the extent of homologyto previously identified sequence and/or structure landmarks. Cloningand expression of a polypeptide-encoding

region of a nucleic acid molecule provides a polypeptide product forstructural and functional analysis. Manipulation of nucleic acidmolecules and encoded polypeptides to produce variants and derivativesthereof may confer advantageous properties on a product for use as atherapeutic.

However, in spite of the significant technical advances in genomeresearch over the past decade, the potential for development of noveltherapeutics based on the human genome is still largely unrealized.While a number of genes encoding potentially beneficial proteintherapeutics, or those encoding polypeptides which may act as “targets”for therapeutic molecules, have been identified using recombinant DNAtechnology, the structure and function of a vast number of genes in thegenome of mammals are yet unknown.

Identification and Characterization of TNF-Family of Ligands andReceptors

Tumor necrosis factor (TNF) was first identified in the serum of miceand rabbits which had been infected with bacillus of Calmette and Guerin(BCG) and which had been injected with endotoxin. TNF activity in theserum of these animals was recognized on the basis of its cytotoxic andanti-tumor activities. This TNF activity, referred to as TNF-α, isproduced particularly by activated monocytes and macrophages, and hasbeen implicated in normal growth processes as well as in a variety ofdiseases.

Following the discovery of TNF-α, independent research led to theidentification of another cytokine associated with inflammatoryresponses lymphotoxin-α (LT-α), which was shown to be producedexclusively by lymphocytes. LT-α was subsequently shown to be 30%homologous with TNF-α, and was renamed TNF-β. It is now clear that TNF-αand TNF-β are members of a gene family that includes yet another membertermed LT-β (Browning et al., Cell 72:847-856, 1993). The three genesare tightly linked within the MHC complex and show similar organization.Moreover, the biologically active forms of TNF-α and TNF-β arehomotrimers and share many of the same biological activities includingcompeting for the same cell-surface receptors (Agarwal et al., Nature318:665-667, 1985). Two distinct but structurally homologous receptorshave been identified, and each has been shown to bind both the ligandsand mediate their effects.

However, it has been recognized that TNFs are only representativemembers of the rapidly expanding supergene familiy that includes TNF-α,TNF-β/lymphotoxin-α (LT-α), lymphotoxin-β (LT-β), FasL, CD40L, CD30L,CD27L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), RANKL,GITRL and TNF-2. See generally Orlinick et al., Cell Signal,10(8):543-551 (1998). The distinctive but overlapping cellular responsesinduced by members of the TNF family of ligands following theirinteraction(s) with their cognate cell-surface receptors result inclearly defined developmental and regulatory changes in cells of thelymphoid, hematopoietic, and other lineages. For example, the TNF familyof ligands are involved in growth regulation and differentiation ofcells which are involved in inflamation, immune processes andhematopoiesis (Bayert, R. and Fiers, W., Tumor Necrosis Factor andLymphokines in: Cytokines eds. Anthony Mire-Sluis and Robin Thorpe,Academic Press San Diego Calif., 1998). The TNF family of ligandsactivates the immune defenses against parasites, and act directly orindirectly as mediators in immune reactions and inflammatory processes.However, administration of TNF and/or other members of the TNF familycan also be accompanied by harmful phenomena such as shock and tissuedamage (Bayert, R. and Fiers, W., supra). The main physiological role ofthe TNF family of ligands is the activation of first-line reaction of anorganism to microbial, parasitic, viral infections, or to mechanicalstress and cancer. For example, TNF-related apoptosis-inducing ligand(TRAIL) has been demonstrated to induce apoptosis of a number ofdifferent types of cancer cells as well as virally infected cells.

Furthermore, a number of observations have also led to the conclusionthat the TNF family of ligands are also involved in a variety ofpathological conditions including cachexia, toxic shock syndrome,inflammatory diseases such as rheumatoid and osteoarthritis, and inlethality resulting from graft-versus-host reaction (GVHR) rapidnecrosis of tumors, apoptosis, immunostimulation and resistance toparasites and viruses. (Bayert, R. and Fiers, W., supra).

Like other cytokines, the members of the TNF family of ligands act viaspecific cell-surface receptors. The receptors, with two exceptions, aretype 1 membrane-associated proteins. Sequence homology amongst them isalmost entirely confined to their extracellular domains. For example,two TNF receptors have been cloned which differ in size and in bindingaffinity (Bayert, R. and Fiers, W., supra). Both receptors bind TNF-αand TNF-β and are membrane associated proteins. The two receptorsconsist of extracellular domains which bind TNF (and are homologous for28%), single transmembrane domains, and intracellular domains which aretotally different from each other and which do not contain anyrecognizable structural motifs that have been associated with anyparticular function. Based on similarities in their extracellulardomains, these receptors belong to a receptor gene superfamily thatincludes the low-affinity nerve growth factor (NGF) receptor, the Fasantigen, the human B-lymphocyte activation molecule CD40, CD27, 4-1BB,PV-T2, CD30, TNFR-RP, TRAIL-R, PV-A53R, RANK, GITR, and the OX40 antigenfound on activated T-cells (Smith et al., Cell, 76(6):959-962, 1994;Baker and Reddy, Oncogene, 12(1):1-9, 1996).

In addition to the membrane associated receptor molecules describedabove, a number the receptors belonging to the TNF-receptor supergenefamily exist as soluble ligand binding proteins. Many of the solubleforms of the transmembrane receptors were subsequently identified ascontaining only the extracellular ligand binding domain(s) of thereceptors. For example, a soluble form of TNF receptor has been found inurine and serum (See U.S. Pat. No. 5,843,789 and Nophar et al., EMBO J.,9(10):3269-3278, 1990), and has been shown to arise by proteolyticcleavage of cell surface TNF-receptors (Wallach et al., Agents ActionsSuppl., 35:51-57 1991). These soluble forms of receptor molecules havebeen implicated in the modulation of TNF activity by not onlyinterfering with TNF binding to its receptor, but also by stabilizingthe TNF structure and preserving its activity, thus prolonging some ofits effects (Aderka et al, Cytokine & Growth Factor Reviews,7(3):231-240, 1996).

The activity of members of the TNF family of ligands is tightlyregulated at the levels of secretion and receptor expression. Additionalregulatory mechanisms are provided by action of specific inhibitoryproteins present on cell surfaces and in biological fluids. While someof these inhibitory proteins have been identified as soluble forms ofreceptor molecules, the identity of many of these cytokine regulatoryproteins are as yet unknown. However, abnormalities in the production ofthese substances might contribute to the pathophysiology of a variety ofdiseases including immune and neoplastic diseases. Besides their role inregulating cytokine activity in vivo, these regulatory molecules holdsignificant potential for therapeutic use as very specificinhibitors/anti-cytokine agents, and as indicators in diagnosis andassessment of immune function and growth parameters in a variety ofautoimmune and malignant diseases.

Because of the important role of the TNF family of ligands (and theirreceptors) in health and disease, a need exists to identify, isolate,and characterize additional members of the family, for use in diagnosingand treating disease and pathological conditions.

SUMMARY OF THE INVENTION

The present invention relates to a novel serine/threonine kinase familyand uses thereof. More specifically, the present invention relates tonovel Fhm nucleic acid molecules and encoded polypeptides, and usesthereof.

The invention provides for an isolated nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of:

-   -   (a) the nucleotide sequence set forth in SEQ ID NO: 3;    -   (b) a nucleotide sequence encoding the polypeptide set forth in        SEQ ID NO: 4;    -   (c) a nucleotide sequence which hybridizes under moderately or        highly stringent conditions to the complement of (a) or (b),        wherein the encoded polypeptide has an activity of the        polypeptide set forth in SEQ ID NO: 3; and    -   (d) a nucleotide sequence complementary to any of (a) through        (c).

The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

-   -   (a) a nucleotide sequence encoding a polypeptide that is at        least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent        identical to the polypeptide set forth in 4, wherein the        polypeptide has an activity of the encoded polypeptide set forth        in SEQ ID NO: 4 as determined using a computer program selected        from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA,        BLASTX, BestFit, and the Smith-Waterman algorithm;    -   (b) a nucleotide sequence encoding an allelic variant or splice        variant of the nucleotide sequence set forth in SEQ ID NO: 3,        wherein the encoded polypeptide has an activity of the        polypeptide set forth in SEQ ID NO: 4;    -   (c) a nucleotide sequence of SEQ ID NO: 3, (a), or (b) encoding        a polypeptide fragment of at least about 25 amino acid residues,        wherein the polypeptide has an activity of the polypeptide set        forth in SEQ ID NO: 4;    -   (d) a nucleotide sequence encoding a polypeptide that has a        substitution and/or deletion of 1 to 251 amino acid residues set        forth in any of SEQ ID NOS: 3-4 wherein the encoded polypeptide        has an activity of the polypeptide set forth in SEQ ID NO: 4;    -   (e) a nucleotide sequence of SEQ ID NO: 3, or (a)-(d) comprising        a fragment of at least about 16 nucleotides;    -   (f) a nucleotide sequence which hybridizes under moderately or        highly stringent conditions to the complement of any of (a)-(e),        wherein the encoded polypeptide has an activity of the        polypeptide set forth in SEQ ID NO:4; and    -   (g) a nucleotide sequence complementary to any of (a)-(e).

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

-   -   (a) a nucleotide sequence encoding a polypeptide set forth in        SEQ ID NO: 4 with at least one conservative amino acid        substitution, wherein the encoded polypeptide has an activity of        the polypeptide set forth in SEQ ID NO: 4;    -   (b) a nucleotide sequence encoding a polypeptide set forth in        SEQ ID NO: 4 with at least one amino acid insertion, wherein the        encoded polypeptide has an activity of the polypeptide set forth        in SEQ ID NO: 4;    -   (c) a nucleotide sequence encoding a polypeptide set forth in        SEQ ID NO: 4 with at least one amino acid deletion, wherein the        encoded polypeptide has an activity of the polypeptide set forth        in SEQ ID NO: 4;    -   (d) a nucleotide sequence encoding a polypeptide set forth in        SEQ ID NO: 4 which has a C- and/or N-terminal truncation,        wherein the encoded polypeptide has an activity of the        polypeptide set forth in SEQ ID NO: 4;    -   (e) a nucleotide sequence encoding a polypeptide set forth in        SEQ ID NO: 4 with at least one modification selected from the        group consisting of amino acid substitutions, amino acid        insertions, amino acid deletions, C-terminal truncation, and        N-terminal truncation, wherein the polypeptide has an activity        of the encoded polypeptide set forth in SEQ ID NO: 4;    -   (f) a nucleotide sequence of (a)-(e) comprising a fragment of at        least about 16 nucleotides;    -   (g) a nucleotide sequence which hybridizes under moderately or        highly stringent conditions to the complement of any of (a)-(f),        wherein the encoded polypeptide has an activity of the        polypeptide set forth in SEQ ID NO: 4; and    -   (h) a nucleotide sequence complementary to any of (a)-(e).

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

-   -   (a) the mature amino acid sequence set forth in SEQ ID NO: 4        comprising a mature amino terminus at residue 1, and optionally        further comprising an amino-terminal methionine;    -   (b) an amino acid sequence for an ortholog of SEQ ID NO: 4,        wherein the polypeptide has an activity of the polypeptide set        forth in SEQ ID NO: 4;    -   (c) an amino acid sequence that is at least about 70, 75, 80,        85, 90, 95, 96, 97, 98, or 99 percent identical to the amino        acid sequence of SEQ ID NO: 4, wherein the polypeptide has an        activity of the polypeptide set forth in SEQ ID NO: 4 as        determined using a computer program selected from the group        consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX,        BestFit, and the Smith-Waterman algorithm;    -   (d) a fragment of the amino acid sequence set forth in SEQ ID        NO: 4 comprising at least about 25 amino acid residues, wherein        the polypeptide has an activity of the polypeptide set forth in        SEQ ID NO: 4;    -   (e) an amino acid sequence for an allelic variant or splice        variant of either the amino acid sequence set forth in SEQ ID        NO: 4, or at least one of (a)-(c) wherein the polypeptide has an        activity of the polypeptide set forth in SEQ ID NO: 4.

The invention further provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

-   -   (a) the amino acid sequence set forth in SEQ ID NO: 4 with at        least one conservative amino acid substitution, wherein the        polypeptide has an activity of the polypeptide set forth in SEQ        ID NO: 4;    -   (b) the amino acid sequence set forth in SEQ ID NO: 4 with at        least one amino acid insertion, wherein the polypeptide has an        activity of the polypeptide set forth in SEQ ID NO: 4;    -   (c) the amino acid sequence set forth in SEQ ID NO: 4 with at        least one amino acid deletion, wherein the polypeptide has an        activity of the polypeptide set forth in SEQ ID NO: 4;    -   (d) the amino acid sequence set forth in SEQ ID NO: 4 which has        a C- and/or N-terminal truncation, wherein the polypeptide has        an activity of the polypeptide set forth in SEQ ID NO: 4; and    -   (e) the amino acid sequence set forth in SEQ ID NO: 4, with at        least one modification selected from the group consisting of        amino acid substitutions, amino acid insertions, amino acid        deletions, C-terminal truncation, and N-terminal truncation,        wherein the polypeptide has an activity of the polypeptide set        forth in SEQ ID NO: 4.

Also provided are fusion polypeptides comprising the polypeptidesequences of (a)-(e) above of the preceding paragraphs.

The present invention also provides for an expression vector comprisingthe isolated nucleic acid molecules set forth herein, recombinant hostcells comprising recombinant nucleic acid molecules set forth herein,and a method of producing a Fhm polypeptide comprising culturing thehost cells and optionally isolating the polypeptide so produced.

A transgenic non-human animal comprising a nucleic acid moleculeencoding a Fhm polypeptide is also encompassed by the invention. The Fhmnucleic acid molecules are introduced into the animal in a manner thatallows expression and increased levels of the Fhm polypeptide, which mayinclude increased circulating levels. The transgenic non-human animal ispreferably a mammal.

Also provided are derivatives of the Fhm polypeptides of the presentinvention.

Additionally provided are selective binding agents such as antibodiesand peptides capable of specifically binding the Fhm polypeptides of theinvention. Such antibodies and peptides may be agonistic orantagonistic.

Pharmaceutical compositions comprising the nucleotides, polypeptides, orselective binding agents of the present invention and one or morepharmaceutically acceptable formulation agents are also encompassed bythe invention. The pharmaceutical compositions are used to providetherapeutically effective amounts of the nucleotides or polypeptides ofthe present invention. The invention is also directed to methods ofusing the polypeptides, nucleic acid molecules and selective bindingagents. The invention also provides for devices to administer a Fhmpolypeptide encapsulated in a membrane.

The Fhm polypeptides and nucleic acid molecules of the present inventionmay be used to treat, prevent, ameliorate, diagnose and/or detectdiseases and disorders, including those recited herein. Expressionanalysis in biological, cellular or tissue samples suggests that Fhmpolypeptide may play a role in the diagnosis and/or treatment ofTNF-related diseases including, but not limited to,acquired-immunodeficiency syndrome (AIDS), anemia, autoimmune diseases,cachexia, cancer, cerebral malaria, diabetes mellitus, disseminatedintravascular coagulopathy, erythryoid sick syndrome, hemorrhagic shock,hepatitis, insulin resistance, leprosy, leukemia, lymphoma, meningitis,multiple sclerosis, myocardial ischaemia, obesity, rejection oftransplanted organs, rheumatoid arthritis, septic shock syndrome,stroke, adult respiratory distress syndrome (ARDS), tuberculosis, and anumber of viral diseases. This expression can de detected with adiagnostic agent such as Fhm nucleotide.

The invention encompasses diagnosing a pathological condition or asusceptibility to a pathological condition in a subject caused by orresulting from abnormal levels of Fhm polypeptide comprising determiningthe presence or amount of expression of the Fhm polypeptide in a sample;and comparing the level of said polypeptide in a biological, tissue orcellular sample from either normal subjects or the subject at an earliertime, wherein susceptibility to a pathological condition is based on thepresence or amount of expression of the polypeptide.

The present invention also provides a method of assaying test moleculesto identify a test molecule which binds to a Fhm polypeptide. The methodcomprises contacting a Fhm polypeptide with a test molecule and todetermine the extent of binding of the test molecule to the polypeptide.The method further comprises determining whether such test molecules areagonists or antagonists of a Fhm polypeptide. The present inventionfurther provides a method of testing the impact of molecules on theexpression of Fhm polypeptide or on the activity of Fhm polypeptide.

Methods of regulating expression and modulating (i.e., increasing ordecreasing) levels of a Fhm polypeptide are also encompassed by theinvention. One method comprises administering to an animal a nucleicacid molecule encoding a Fhm polypeptide. In another method, a nucleicacid molecule comprising elements that regulate or modulate theexpression of a Fhm polypeptide may be administered. Exampless of thesemethods include gene therapy, cell therapy, and anti-sense therapy asfurther described herein.

Surprisingly, a Fhm polypeptide was highly expressed in a wide range ofprimary human tumors. Therefore, the present polypeptide, and its usefulnucleid acid intermediates, have demonstrated utility in differentiatingtransformed cells from the background.

In another aspect of the present invention, the Fhm polypeptides may beused for identifying receptors or binding partners thereof (“Fhmreceptors” or “Fhm binding partners”). Various forms of “expressioncloning” have been extensively used to clone receptors for protein orco-factors. See, for example, Simonsen and Lodish, Trends inPharmacological Sciences, 15: 437-441, 1994, and Tartaglia et al., Cell,83:1263-1271, 1995. The isolation of the Fhm receptor(s) or Fhm bindingpartner(s) is useful for identifying or developing novel agonists andantagonists of the Fhm polypeptide-signaling pathway.

Such agonists and antagonists include soluble Fhm ligand(s), anti-Fhmselective binding agents (such as Fhm antibodies and derivativesthereof), small molecules, peptides or derivatives thereof capable ofbinding Fhm polypeptides, or antisense oligonucleotides, any of whichcan be used for potentially treating one or more diseases or disorders,including those recited herein.

In certain embodiments, a Fhm polypeptide agonist or antagonist may be aprotein, peptide, carbohydrate, lipid, or small molecular weightmolecule which interacts with Fhm polypeptide to regulate its activity.

In another aspect of the present invention, the Fhm polypeptides may beused for identifying receptors thereof (“Fhm receptors”). Various formsof “expression cloning” have been extensively used for cloning to clonereceptors for protein ligands. See for example, H. Simonsen and H. F.Lodish, Trends in Pharmacological Sciences, vol. 15, 437-44115:437-44,1994, and Tartaglia et al., Cell, 83:1263-1271, 1995. The isolation ofthe Fhm receptor(s) is useful for identifying or developing novelagonists and antagonists of the Fhm polypeptide-signaling pathway. Suchagonists and antagonists include soluble Fhm receptor(s), anti-Fhmreceptor selectivereceptor-selective binding agents (such as antibodiesand derivatives thereof), small molecules, and antisenseoligonucleotides, any of which can be used for treating one or more ofthe diseases or disorders, including those recited herein.

DESCRIPTION OF THE FIGURE

FIGS. 1A-1B present an alignment of the predicted amino acid sequence ofFhm polypeptide (SEQ ID NO: 4) is aligned with the corresponding regionsof human FasL, mouse FasL, rat FasL, human CD40L, mouse CD40L, mouseOPGL, human OPGL, human TRAIL, mouse TRAIL, human CD30L, human CD30L,human LyT-β, mouse LyT-β, human TNF-β, mouse TNF-β, human TNF-α andmouse TNF-α. (SEQ ID NOS: 5-21) using the Pileup program (Wisconsin GCGProgram Package ver. 8.1).

DETAILED DESCRIPTION OF THE INVENTION

The section headings herein are for organizational purposes only and arenot to be construed as limiting the subject matter described therein.

Definitions:

The terms “Fhm gene”, “Fhm nucleic acid molecule”, or “Fhmpolynucleotide” refer to a nucleic acid molecule comprising orconsisting essentially of a nucleotide sequence as set forth in SEQ IDNO: 3, comprising or consisting essentially of a nucleotide sequenceencoding the polypeptide as set forth in SEQ ID NO: 4, or nucleic acidmolecules related thereto. “Related” nucleic acid molecules comprise orconsist essentially of a nucleotide sequence that is about 70 percentidentical to the nucleotide sequence as shown in SEQ ID NO: 3, orcomprise or consist essentially of a nucleotide sequence encoding apolypeptide having an amino acid sequence that is about 70 percentidentical to the amino acid sequence set forth in SEQ ID NO: 4. Inpreferred embodiments, the nucleotide sequences are about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the nucleotide sequence as shownin SEQ ID NO: 3, or the nucleotide sequences encode a polypeptide thatis about 75 percent, or about 80 percent, or about 85 percent, or about90 percent, or about 95, 96, 97, 98, or 99 percent identical to thepolypeptide sequence as set forth in SEQ ID NO: 4. Related nucleic acidmolecules also include fragments of the above Fhm nucleic acid moleculeswhich are at least about 10 contiguous nucleotides, or about 15, orabout 20, or about 25, or about 50, or about 75, or about 100, orgreater than about 100 contiguous nucleotides. Related nucleic acidmolecules also include fragments of the above Fhm nucleic acid moleculeswhich encode a polypeptide of at least about 25 amino acid residues, orabout 50, or about 75, or about 100, or greater than about 100 aminoacid residues. Related nucleic acid molecules also include a nucleotidesequence encoding a polypeptide comprising or consisting essentially ofa substitution and/or a deletion of one to 251 amino acid residuescompared to the polypeptide in SEQ ID NO: 4. Related Fhm ligand nucleicacid molecules include those molecules which comprise nucleotidesequences which hybridize under moderate or highly stringent conditionsas defined herein with any of the above nucleic acid molecules. Inpreferred embodiments, the related nucleic acid molecules comprisesequences which hybridize under moderate or highly stringent conditionswith the sequence as shown in SEQ ID NO:3, or with a molecule encoding apolypeptide, which polypeptide comprises the amino acid sequence asshown in SEQ ID NO:4, or with a nucleic acid fragment as defined above,or with a nucleic acid fragment encoding a polypeptide as defined above.It is also understood that related nucleic acid molecules includeallelic or splice variants of any of the above nucleic acids, andinclude sequences which are complementary to any of the above nucleotidesequences.

The term “Fhm polypeptide allelic variant” refers to one of severalpossible naturally occurring alternate forms of a gene occupying a givenlocus on a chromosome of an organism or a population of organisms.

The term “Fhm polypeptide splice variant” refers to a nucleic acidmolecule, usually RNA, which is generated by alternative processing ofintron sequences in an RNA transcript of Fhm polypeptide amino acidsequence set forth in SEQ ID NO: 4.

The term “expression vector” refers to a vector which is suitable foruse in a host cell and contains nucleic acid sequences which directand/or control the expression of inserted heterologous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

The term “Fhm polypeptide” refers to a polypeptide comprising the aminoacid sequence of SEQ ID NO: 4, and related polypeptides. Relatedpolypeptides includes: Fhm allelic variants, Fhm splice variants, Fhmfragments, Fhm derivatives, Fhm-substitution, -deletion, and/orinsertion variants, Fhm fusion polypeptides, and Fhm orthologs. Fhmpolypeptides may be mature polypeptides, as defined herein, and may ormay not have an amino terminal methionine residue, depending on themethod by which they are prepared.

The term Fhm polypeptide fragment refers to a peptide or polypeptidethat comprises less than the full length amino acid sequence of a Fhmpolypeptide as set forth in SEQ ID NO: 4. Such a fragment may arise, forexample, from a truncation at the amino terminus (with or without aleader sequence), a truncation at the carboxy terminus, and/or aninternal deletion of the amino acid sequence (wherein the resultingpolypeptide is at lease 6 amino acids or more in length). Fhm fragmentsmay result from alternative RNA splicing or from in vivo proteaseactivity.

In preferred embodiments, truncations comprise about 10 amino acids, orabout 20 amino acids, or about 50 amino acids, or about 75 amino acids,or about 100 amino acids, or more than about 100 amino acids. Thepolypeptide fragments so produced will comprise about 25 contiguousamino acids, or about 50 amino acids, or about 75 amino acids, or about100 amino acids, or about 150 amino acids, or about 200 amino acids.Such Fhm polypeptide fragments may optionally comprise an amino terminalmethionine residue. It will be appreciated that such fragments can beused, for example, to generate antibodies to Fhm polypeptides.

The term “Fhm polypeptide variants” refers to Fhm polypeptidescomprising amino acid sequences which contain one or more amino acidsequence substitutions, deletions (such as internal deletions and/or Fhmfragments), and/or additions (such as internal additions and/or Fhmfusion polypeptides) as compared to the Fhm polypeptide amino acidsequence set forth in SEQ ID NO: 4 (with or without leader sequences).Variants may be naturally occurring (e.g., Fhm polypeptide allelicvariants, Fhm polypeptide orthologs or Fhm splice variants) orartificially constructed. Such Fhm-polypeptide variants may be preparedfrom the corresponding nucleic acid molecules encoding said variants,which have a DNA sequence that varies accordingly from the DNA sequencesfor wild type Fhm-receptor polypeptides as set forth in SEQ ID NO: 3.

The term “Fhm fusion polypeptide” “Fhm fusion polypeptide” refers to afusion of one or more amino acids (such as a heterologous peptide orpolypeptide) at the amino or carboxy terminus of the polypeptide as setforth in SEQ ID NO: 4, Fhm polypeptide allelic variants, Fhm polypeptideorthologs, Fhm polypeptide splice variants, or Fhm polypeptide variantshaving one or more amino acid deletions, substitutions or internaladditions as compared to the Fhm polypeptide amino acid sequence setforth in SEQ ID NO: 4.

The term “Fhm polypeptide derivatives” refers to Fhm polypeptides,variants, or fragments thereof, that have been chemically modified, asfor example, by covalent attachment of one or more water solublepolymers, N-linked or O-linked carbohydrates, sugars, phosphates, and/orother such molecules. Such modifications may be introduced into themolecule by reacting targeted amino acid residues of the purified orcrude protein with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. The resultingcovalent derivatives are also useful in programs directed at identifyingresidues important for biological activity. The derivatives are modifiedin a manner that is different from naturally occurring Fhm polypeptideeither in the type or location of the molecules attached to thepolypeptide. Derivatives further include deletion of one or morechemical groups naturally attached to the Fhm polypeptide.

The terms “biologically active Fhm polypeptides”, “biologically activeFhm polypeptide fragments”, “biologically active Fhm polypeptidevariants”, and “biologically active Fhm polypeptide derivatives” referto Fhm polypeptides having at least one activity characteristic of a Fhmpolypeptide comprising the amino acid sequence of SEQ ID NO: 4, such asthe ability to bind to one or more members of the TNF-receptor supergene (protein) family in biological assays. Immunogenic fragments of Fhmpolypeptides are those capable of inducing in a host animal antibodiesdirected to the Fhm fragment.

The term “Fhm polypeptide ortholog” refers to a polypeptide from anotherspecies that corresponds to a Fhm polypeptide amino acid sequence setforth as SEQ ID NO: 4. For example, mouse and human Fhm polypeptides areconsidered orthologs of each other.

The term “mature Fhm polypeptide” refers to a Fhm polypeptide lacking aleader sequence, a mature Fhm polypeptide may also include othermodifications of a polypeptide such as proteolytic processing of theamino terminus (with or without a leader sequence) and/or the carboxyterminus, cleavage of a smaller polypeptide from a larger precursor,N-linked and/or O-linked glycosylation, and the like.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that each antigen. Anantigen may have one or more epitopes.

The term “mutein” refers to a mutant protein, polypeptide, variants,analogs or fragments of Fhm polypeptide. Muteins of Fhm may be preparedby deletion, insertion, substitution, point mutation, truncation,addition, transposition, PCR amplification, site-directed mutagenesis orother methods known in the art.

The terms “effective amount” and “therapeutically effective amount”refer to the amount of a Fhm polypeptide (or Fhm antagonist) necessaryto support an observable change in the level of one or more biologicalactivities of one or more members of the TNF-receptor gene family as setforth above, to bring about a meaningful patient benefit, i.e.treatment, healing, prevention, or amelioration of a condition. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of active ingredients that result intherapeutic effect, when administered in combination, serially orsimultaneously. The Fhm polypeptides that have use in practicing thepresent invention may be naturally occurring full length polypeptides,or truncated polypeptides or variant homologs or analogs or derivativesor peptide fragments. Illustrative analogs include those in which one ormore divergent amino acids between two species are substituted with thedivergent amino acid from another species. Divergent amino acids mayalso be substituted with any other amino acid whether it be aconservative or a non-conservative amino acid.

The term “identity”, as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessnucleic acid molecules or polypeptides sequences, as the case may be, asdetermined by the match between strings of two or more nucleotide or twoor more amino acid sequences. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by particular a mathematical model ofcomputer program (i.e., “algorithms”). The term “similarity” is arelated concept, but in contrast to “identity”, refers to a measure ofsimilarity which includes both identical matches and conservativesubstitution matches. If two polypeptide sequences have, for example,{fraction (10/20)} identical amino acids, and the remainder are allnon-conservative substitutions, then the percent identity and similaritywould both be 50%. If, in the same example, there are 5 more positionswhere there are conservative substitutions, then the percent identityremains 50%, but the percent similarity would be 75% ({fraction(15/20)}). Therefore, in cases where there are conservativesubstitutions, the degree of percent similarity between two polypeptidesequences will be higher than the percent identity between those twosequences.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates or other materialswith which it is naturally found when total DNA is isolated from thesource cells, (2) is not linked to all or a portion of a polynucleotideto which the “isolated” isolated nucleic acid molecule “molecule” islinked in nature, (3) is operably linked to a polynucleotide which it isnot linked to in nature, or (4) does not occur in nature as part of alarger polynucleotide sequence. Preferably, the isolated nucleic acidmolecule of the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates or other materials with which itis naturally found when isolated from the source cell, (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature, (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature, or (4) does not occurin nature. Preferably, the isolated polypeptide is substantially freefrom any other contaminating polypeptides or other contaminants that arefound in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

The terms “nucleic acid sequence” or “nucleic acid molecule” refer to aDNA or RNA sequence. The term encompassesterms encompass moleculesformed from any of the known base analogs of DNA and RNA such as, butnot limited to 4-4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” “non-naturally occurring” or “non-native” as used hereinrefers to a material that is not found in nature or that has beenstructurally modified or synthesized by man.

The term “operably linked” “operably linked” is used herein to refer toan arrangementa method of flanking sequences wherein the flankingsequences so described are configured or assembled so as to performtheir usual function. Thus, a flanking sequence operably linked to acoding sequence may be capable of effecting the replication,transcription and/or translation of the coding sequence. For example, acoding sequence is operably linked to a promoter when the promoter iscapable of directing transcription of that coding sequence. A flankingsequence need not be contiguous with the coding sequence, so long as itfunctions correctly. Thus, for example, intervening untranslated yettranscribed sequences can be present between a promoter sequence and thecoding sequence, and the promoter sequence can still be considered“operably linked” “operably linked” to the coding sequence.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers terms “pharmaceuticallyacceptable carrier” or “physiologically acceptable carrier” as usedherein refer to one or more formulation materials suitable foraccomplishing or enhancing the delivery of the Fhm polypeptide, Fhmnucleic acid molecule or Fhm selective binding agent as a pharmaceuticalcomposition.

The term “selective binding agent” refers to a molecule or moleculeshaving specificity for an Fhm polypeptide. As used herein, the terms,“specific” and “specificity” refer to the ability of the selectivebinding agents to bind to human Fhm polypeptides and not to bind tohuman non-Fhm polypeptides. It will be appreciated, however, that theselective binding agents may also bind orthologs of the polypeptide asset forth in SEQ ID NO: 4, that is, interspecies versions thereof, suchas mouse and rat polypeptides.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, for example, Graham et al., Virology, 52:456, 1973;Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratories, New York, 1989; Davis et al., Basic Methods inMolecular Biology, Elsevier, 1986; and Chu et al., Gene, 13:197, 1981.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, it may be maintained transiently as an episomal element withoutbeing replicated, or I may replicate independently as a plasmid. A cellis considered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

It is understood that related nucleic acid molecules include allelic orsplice variants of the nucleic acid molecule of SEQ ID NO: 3, andinclude sequences which are complementary to any of the above nucleotidesequences. Related nucleic acid molecules also include a nucleotidesequence encoding a polypeptide comprising or consisting essentially ofa substitution, modification, addition and/or a deletion of one or moreamino acid residues compared to the polypeptide in SEQ ID NO: 4.

Fragments include molecules which encode a polypeptide of at least about25 amino acid residues, or about 50, or about 75, or about 100, orgreater than about 100, amino acid residues of the polypeptide of SEQ IDNO: 4.

In addition, related Fhm nucleic acid molecules include those moleculeswhich comprise nucleotide sequences which hybridize under moderately orhighly stringent conditions as defined herein with the fullycomplementary sequence of the nucleic acid molecule of SEQ ID NO: 3, orof a molecule encoding a polypeptide, which polypeptide comprises theamino acid sequence as shown in SEQ ID NO: 4, or of a nucleic acidfragment as defined herein, or of a nucleic acid fragment encoding apolypeptide as defined herein. Hybridization probes may be preparedusing the Fhm sequences provided herein to screen cDNA, genomic orsynthetic DNA libraries for related sequences. Regions of the DNA and/oramino acid sequence of Fhm polypeptide that exhibit significant identityto known sequences are readily determined using sequence alignmentalgorithms as described herein, and those regions may be used to designprobes for screening.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, (Cold Spring Harbor, N.Y. 1989); and Anderson et al.,Nucleic Acid Hybridization: a Practical approach, Ch. 4, IRL PressLimited (Oxford, England). Limited, Oxford, England. More stringentconditions (such as higher temperature, lower ionic strength, higherformamide, or other denaturing agent) may also be used, used; however,the rate of hybridization will be affected. Other agents may be includedin the hybridization and washing buffers for the purpose of reducingnon-specific and/or background hybridization. Examples are 0.1% bovineserum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate,0.1% sodium dodecylsulfate (NaDodSO4 or SDS), ficoll, Denhardt'ssolution, sonicated salmon sperm DNA (or another non-complementary DNA),and dextran sulfate, although other suitable agents can also be used.The concentration and types of these additives can be changed withoutsubstantially affecting the stringency of the hybridization conditions.Hybridization experiments are usually carried out at pH 6.8-7.4,6.8-7.4; however, at typical ionic strength conditions, the rate ofhybridization is nearly independent of pH. See Anderson et al., NucleicAcid Hybridization: a Practical Approach, Ch. 4, IRL Press Limited(Oxford, England).

Factors affecting the stability of a DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:

 Tm(° C.)=81.5+16.6(log[Na+])+0.41(% G+C)−600/N−0.72(% formamide)

where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately” stringent conditions” “refers to conditions underwhich a DNA duplex with a greater degree of base pair mismatching thancould occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, a “moderately stringent” condition of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature orraise the ionic strength.

A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20 nt is given by:Tm=2° C. per A−T base pair+4° C. per G−C base pair*The sodium ion concentration in 6×salt sodium citrate (SSC) is 1 M. SeeSuggs et al., Developmental Biology Using Purified Genes, p. 683, Brownand Fox (eds.) (1981). High stringency washing conditions foroligonucleotides are usually at a temperature of 0-5° C. below the Tm ofthe oligonucleotide in 6×SSC, 0.1% SDS.

In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is about 70 percent (70%)identical to the nucleotide sequence as shown in SEQ ID NO: 3, orcomprise or consist essentially of a nucleotide sequence encoding apolypeptide that is about 70 percent (70%) identical to the polypeptideas set forth in SEQ ID NO: 4. In preferred embodiments, the nucleotidesequences are about 75 percent, or about 80 percent, or about 85percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percentidentical to the nucleotide sequence as shown in SEQ ID NO: 3, or thenucleotide sequences encode a polypeptide that is about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the polypeptide sequence as setforth in SEQ ID NO: 4.

Differences in the nucleic acid sequence may result in conservativeand/or non-conservative modifications of the amino acid sequencerelative to the amino acid sequence of SEQ ID NO: 4.

Conservative modifications to the amino acid sequence of SEQ ID NO: 4(and corresponding modifications to the encoding nucleotides) willproduce Fhm polypeptides having functional and chemical characteristicssimilar to those of a naturally occurring Fhm polypeptide. In contrast,substantial modifications in the functional and/or chemicalcharacteristics of Fhm polypeptides may be accomplished by selectingsubstitutions in the amino acid sequence of SEQ ID NO: 4 that differsignificantly in their effect on maintaining (a) the structure of themolecular backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” “conservativeamino acid substitution” may involve a substitution of a native aminoacid residue with a normative residue such that there is little or noeffect on the polarity or charge of the amino acid residue at thatposition. Furthermore, any native residue in the polypeptide may also besubstituted with alanine, as has been previously described for “alaninescanning mutagenesis.”

Naturally occurring residues may be divided into groups based on commonside chain properties:

-   -   1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   2) neutral hydrophilic: Cys, Ser, Thr;    -   3) acidic: Asp, Glu;    -   4) basic: Asn, Gln, His, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human Fhm polypeptidethat are homologous with non-human Fhm polypeptide orthologs, or intothe non-homologous regions of the molecule.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131, 1982. It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); (−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the Fhmpolypeptide, or to increase or decrease the affinity of the Fhmpolypeptides described herein.

Exemplary amino acid substitutions are set forth in Table I.

TABLE I Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe LysArg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth in SEQ ID NO: 4 using well known techniques.For identifying suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of an Fhm polypeptide to such similar polypeptides. With such acomparison, one can identify residues and portions of the molecules thatare conserved among similar polypeptides. It will be appreciated thatchanges in areas of an Fhm polypeptide that are not conserved relativeto such similar polypeptides would be less likely to adversely affectthe biological activity and/or structure of the Fhm polypeptide. Oneskilled in the art would also know that, even in relatively conservedregions, one may substitute chemically similar amino acids for thenaturally occurring residues while retaining activity (conservativeamino acid residue substitutions). Therefore, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in an Fhm polypeptide thatcorrespond to amino acid residues that which are important for activityor structure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of Fhm polypeptides.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an Fhm polypeptide withrespect to its three dimensional structure. One skilled in the art maychoose not to make radical changes to amino acid residues predicted tobe on the surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. The variants can thenbe screened using activity assays know to those skilled in the art. Suchvariants could be used to gather information about suitable variants.For example, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. (See Moult J., Curr. Op. in Biotech.,7(4):422-427, 1996. Chou et al., Biochemistry, 13(2):222-245, 1974; Chouet al., Biochemistry, 113(2):211-222, 1974; Chou et al., Adv. Enzymol.Relat. Areas Mol. Biol., 47:45-148, 1978; Chou et al., Ann. Rev.Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384, 1979).Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247, 1999. It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376, 1997) that thereare a limited number of folds in a given polypeptide or protein and thatonce a critical number of structures have been resolved, structuralprediction will become dramatically in more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87, 1997; Sippl et al.,Structure, 4(1):15-9 1996), “profile analysis” (Bowie et al., Science,253:164-170, 1991); Gribskov et al., Meth. Enzym., 183:146-159, 1990;Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 1987), and“evolutionary linkage” (See Home, supra, and Brenner, supra).

Preferred Fhm polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites has been alteredcompared to the amino acid sequence set forth in SEQ ID NO: 4. In oneembodiment, Fhm polypeptide variants comprise a greater or a lessernumber of N-linked glycosylation sites than the amino acid sequence setforth in SEQ ID NO: 4. An N-linked glycosylation site is characterizedby the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution(s) of amino acid residues to create this sequence providesa potential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. Also provided is arearrangement of N-linked carbohydrate chains wherein one or moreN-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated.

Additional preferred Fhm variants include cysteine variants, wherein oneor more cysteine residues are deleted from or substituted for anotheramino acid (e.g., serine) as compared to the amino acid sequence setforth in SEQ ID NO: 4. Cysteine variants are useful when Fhmpolypeptides must be refolded into a biologically active conformationsuch as after the isolation of insoluble inclusion bodies. Cysteinevariants generally have fewer cysteine residues than the native protein,and typically have an even number to minimize interactions resultingfrom unpaired cysteines.

In addition, the polypeptide comprising the amino acid sequence of SEQID NO: 4 or an Fhm polypeptide variant may be fused to a homologouspolypeptide to form a homodimer or to a heterologous polypeptide to forma heterodimer. Heterologous peptides and polypeptides include, but arenot limited to: an epitope to allow for the detection and/or isolationof an Fhm fusion polypeptide; a transmembrane receptor protein or aportion thereof, such as an extracellular domain, or a transmembrane andintracellular domain; a ligand or a portion thereof which binds to atransmembrane receptor protein; an enzyme or portion thereof which iscatalytically active; a polypeptide or peptide which promotesoligomerization, such as a leucine zipper domain; a polypeptide orpeptide which increases stability, such as an immunoglobulin constantregion; and a polypeptide which has a therapeutic activity differentfrom the polypeptide comprising the amino acid sequence as set forth inSEQ ID NO: 4 or an Fhm polypeptide variant.

Fusions can be made either at the amino terminus or at the carboxyterminus of the polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 4 or an Fhm polypeptide variant. Fusions may be directwith no linker or adapter molecule, or indirect using a linker oradapter molecule. A linker or adapter molecule may be one or more aminoacid residues, typically up to from about 20 to about 50 amino acidresidues. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for a protease toallow for the separation of the fused moieties. It will be appreciatedthat once constructed, the fusion polypeptides can be derivatizedaccording to the methods described herein.

In a further embodiment of the invention, the polypeptide comprising theamino acid sequence of SEQ ID NO: 4 or an Fhm polypeptide variant isfused to one or more domains of an Fc region of human IgG. Antibodiescomprise two functionally independent parts, a variable domain known as“Fab”, which binds antigens, and a constant domain known as “Fc”, whichis involved in effector functions such as complement activation andattack by phagocytic cells. An Fc has a long serum half-life, whereas anFab is short-lived. (Capon et al., Nature, 337:525-31, 1989). Whenconstructed together with a therapeutic protein, an Fc domain canprovide longer half-life or incorporate such functions as Fc receptorbinding, protein A binding, complement fixation and perhaps evenplacental transfer. Id. Table II summarizes the use of certain Fcfusions known in the art.

TABLE II Fc Fusion with Therapeutic Proteins Therapeutic Form of FcFusion partner implications Reference IgG1 N-terminus of Hodgkin's U.S.Pat. No. CD30-L disease; 5,480,981 anaplastic lymphoma; T- cell leukemiaMurine Fcg2a IL-10 anti- Zheng et al. inflammatory; (1995), J.transplant Immunol., 154: rejection 5590-5600 IgG1 TNF receptor septicshock Fisher et al. (1996), N. Engl. J. Med., 334: 1697- 1702; Van Zeeet al., (1996), J. Immunol., 156: 2221-2230 IgG, IgA, IgM, TNF receptorinflammation, U.S. Pat. No. or IgE autoimmune 5,808,029, issued(excluding the disorders Sep. 15, first domain) 1998 IgG1 CD4 receptorAIDS Capon et al. (1989), Nature 337: 525-531 IgG1, N-terminusanti-cancer, Harvill et al. IgG3 of IL-2 antiviral (1995), Immunotech.,1: 95-105 IgG1 C-terminus of osteoarthritis; WO 97/23614, OPG bonedensity published Jul. 3, 1997 IgG1 N-terminus of anti-obesity PCT/USleptin 97/23183, filed Dec. 11, 1997 Human Ig Cg1 CTLA-4 autoimmuneLinsley (1991), J. disorders Exp. Med., 174: 561-569

In one example, all or a portion of the human IgG hinge, CH2 and CH3regions may be fused at either the N-terminus or C-terminus of the Fhmpolypeptides using methods known to the skilled artisan. The resultingFhm fusion polypeptide may be purified by use of a Protein A affinitycolumn. Peptides and proteins fused to an Fc region have been found toexhibit a substantially greater half-life in vivo than the unfusedcounterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time,reduce aggregation, etc.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J.Applied Math., 48:1073, 1988.

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nucl. Acid. Res., 12:387, 1984; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol., 215:403-410, 1990). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3×the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually {fraction (1/10)} times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. A standard comparison matrix (see Dayhoff et al.,Atlas of Protein Sequence and Structure, vol. 5, supp.35(3), 1978 forthe PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. SciUSA, 89:10915-10919, 1992 for the BLOSUM 62 comparison matrix) is alsoused by the algorithm.

Preferred parameters for polypeptide sequence comparison include thefollowing:

-   -   Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453        (1970),    -   Comparison matrix: BLOSUM 62 from Henikoff and Henikoff, Proc.        Natl. Acad. Sci. USA89:10915-10919 (1992).    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisoninclude the following:

-   -   Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-4, 1970    -   Comparison matrix: matches=+10, mismatch=0    -   Gap Penalty: 50    -   Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version 9, September, 1997. Theparticular choices to be made will be apparent to those of skill in theart and will depend on the specific comparison to be made, such asDNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally,whether the comparison is between pairs of sequences (in which case GAPor BestFit are generally preferred) or between one sequence and a largedatabase of sequences (in which case FASTA or BLASTA are preferred).

Synthesis

It will be appreciated by those skilled in the art the nucleic acid andpolypeptide molecules described herein may be produced by recombinantand other means.

Nucleic Acid Molecules

The nucleic acid molecules encode a polypeptide comprising the aminoacid sequence of an Fhm polypeptide and can readily be obtained in avariety of ways including, without limitation, chemical synthesis, cDNAor genomic library screening, expression library screening and/or PCRamplification of cDNA

Recombinant DNA methods used herein are generally, those set forth inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and/or Ausubelet al., eds., (Current Protocols in Molecular Biology, Green PublishersInc. and Wiley and Sons, N.Y. (1994)).

The present invention provides for nucleic acid molecules as describedherein and methods for obtaining such molecules. Where a gene encodingFhm polypeptide has been identified from one species, all or a portionof that gene may be used as a probe to identify orthologs or relatedgenes from the same species. The probes or primers may be used to screencDNA libraries from various tissue sources believed to express the Fhmpolypeptide

In addition, part or all of a nucleic acid molecule having the sequenceas set forth in SEQ ID NO: 3 may be used to screen a genomic library toidentify and isolate a gene encoding Fhm. Typically, conditions ofmoderate or high stringency will be employed for screening to minimizethe number of false positives obtained from the screen.

Nucleic acid molecules encoding the amino acid sequence of Fhmpolypeptides may also be identified by expression cloning, which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingof an antibody or other binding partner (e.g., receptor or ligand) tocloned proteins which are expressed and displayed on a host cellsurface. The antibody or binding partner is modified with a detectablelabel to identify those cells expressing the desired clone.

Recombinant expression techniques conducted in accordance with thedescriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence which encodes the amino acid sequenceof an Fhm polypeptide into an appropriate vector, one skilled in the artcan readily produce large quantities of the desired nucleotide sequence.The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of an Fhm polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded Fhm polypeptide may be produced in largeamounts.

Another method for obtaining a suitable nucleic acid sequence is thepolymerase chain reaction (PCR). In this method, cDNA is prepared frompoly(A)+RNA or total RNA using the enzyme reverse transcriptase. Twoprimers, typically complementary to two separate regions of cDNA(oligonucleotides) encoding the amino acid sequence of an Fhmpolypeptide, are then added to the cDNA along with a polymerase such asTaq polymerase, and the polymerase amplifies the cDNA region between thetwo primers.

Another means of preparing a nucleic acid molecule encoding the aminoacid sequecne of Fhm polypeptide is by chemical synthesis using methodswell known to the skilled artisan such as those described by Engels etal., Angew. Chem. Intl. Ed., 28:716-734, 1989. These methods include,inter alia, the phosphotriester, phosphoramidite, and H-phosphonatemethods for nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry. Typically, the DNA encoding the amino acid sequence of a Fhmpolypeptide will be several hundred nucleotides in length. Nucleic acidslarger than about 100 nucleotides can be synthesized as severalfragments using these methods. The fragments can then be ligatedtogether to form the full-length nucleotide sequence of a Fhmpolypeptide. Usually, the DNA fragment encoding the amino terminus ofthe polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of the Fhmpolypeptide, depending on whether the polypeptide produce din the hostcell is designed to be secreted from the cell. Other methods known tothe skilled artisan may be used as well.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for the optimal expression of a Fhm polypeptide in a givenhost cell. Particular codon alterations will depend upon the Fhmpolypeptide(s) and host cell(s) selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Ecohigh.cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.Other useful codon frequency tables include “Celegans_high.cod”,“Celegans_low.cod”, “Drosophila_high.cod”, “Human_high.cod”,“Maize_high.cod”, and “Yeast_high.cod”.

In other embodiments, nucleic acid molecules encode Fhm variants withconservative amino acid substitutions as defined above, Fhm variantscomprising an addition and/or a deletion of one or more N-linked orO-linked glycosylation sites, or Fhm polypeptide fragments as describedabove. In addition, nucleic acid molecules may encode any combination ofFhm variants, fragments, and fusion polypeptides described hereinprovided that DNA's modified in this way code for polypeptides capableof finding one or more members of TNF supergene family of ligands andreceptors.

Vectors and Host Cells

A nucleic acid molecule encoding the amino acid sequence of a Fhmpolypeptide may be inserted into an appropriate expression vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of a Fhm polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems),and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether the Fhm polypeptide is to be post-translationallymodified (e.g., glycosylated and/or phosphorylated). If so, yeast,insect, or mammalian host cells are preferable. For a review ofexpression vectors see Meth. Enz. vol. 185 D. V. Goeddel ed., AcademicPress, Sna Diego Calif., 1990.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” (in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forsecretion, a ribosome binding site, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding thepolypeptide to be expressed, and a selectable marker element. Each ofthese sequences is discussed below.

Optionally, the vector may contain a “tag” sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the Fhmpolypeptide coding sequence; the oligonucleotide molecule encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus) or myc for which commercially availableantibodies exist. Optionally, the Fhm gene can also be fused in frame atthe N-terminal for example to an IgG Fc region. This tag is typicallyfused to the polypeptide upon expression of the polypeptide, and canserve as a means for affinity purification of the Fhm polypeptide fromthe host cell although it may also prolong the circulatory half life ofa Fhm polypeptide. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified Fhm polypeptide by various means such as using certainpeptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e, from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), or synthetic, ortehflanking sequence may be native sequences which normally function toregulate Fhm expression. As such, the source of flanking sequences maybe any prokaryotic or eukaryotic organism, any vertebrate orinvertebrate organism, or any plant, provided that a flanking sequenceis functional in, and can be activated by, the host cell machinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein other than the sequences flanking theFhm gene will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species.

Where the flanking sequence is not known, a fragment of DNA containing aflanking sequence may be isolated from a larger piece of DNA that maycontain, for example, a coding sequence or even another gene or genes.Isolation may be accomplished by restriction endonuclease digestion toproduce the proper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother method known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases be important for the optimalexpression of the Fhm polypeptide. If the vector of choice does notcontain an origin of replication site, one may be chemically synthesizedbased on a known sequence, and ligated into the vector. For example, theorigin of replication from the plasmid pBR322 (Product No. 303-3s, NewEngland Biolabs, Beverly, Mass.) is suitable for most Gram-negativebacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV) or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it contains theearly promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding regions and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene which will beexpressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes Fhm. As a result, increased quantities of Fhm aresynthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the Fhm polypeptide to beexpressed. The Shine-Dalgarno sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth herein and used in a prokaryotic vector.

A leader, or signal, sequence may be used to direct the secretion of Fhmpolypeptide out of the host cell where it is synthesized. Typically, anucleotide sequence encoding the signal sequence is positioned in thecoding region of the Fhm nucleic acid molecule, or directly at the 5′end of the Fhm polypeptide coding region. Many signal sequences havebeen identified, and any of those that are functional in the selectedhost cell may be used in conjunction with the Fhm gene or cDNA.Therefore, a signal sequence may be homologous (naturally occurring) orheterologous to the Fhm gene or cDNA, and may be homologous orheterologous to the Fhm gene or cDNA. Additionally, a signal sequencemay be chemically synthesized using methods described herein. In mostcases, the secretion of an Fhm polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the signalpeptide from the Fhm polypeptide.

The signal sequence may be a component of the vector, or it may be apart of Fhm nucleic acid molecule that is inserted into the vector. Thenative Fhm DNA encodes a signal sequence at the amino terminus of theprotein that is cleaved during post-translational processing of themolecule to form the mature Fhm protein product. Included within thescope of this invention are Fhm nucleotides with the native signalsequence as well as Fhm nucleotides wherein the native signal sequenceis deleted and replaced with a heterologous signal sequence. Theheterologous signal sequence selected should be one that is recognizedand processed, i.e., cleaved by a signal peptidase, by the host cell.For prokaryotic host cells that do not recognize and process the nativeFhm signal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, or heat-stable enterotoxin II leaders. Foryeast secretion, the native Fhm signal sequence may be substituted bythe yeast invertase, alpha factor, or acid phosphatase signal sequences.For mammalian cell expression the native signal sequence of the Fhmpolypeptide is satisfactory, although other mammalian signal sequencesmay be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, or addpresequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein), one or more additional amino acid residuesincident to expression, which may not have been totally removed. Forexample, the final protein product may have one or two amino acids foundin the peptidase cleavage site, attached to the N-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired Fhm polypeptide, if the enzymecuts at such area within the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns used may be naturallyoccurring within the Fhm gene, especially where the gene used is a fulllength genomic sequence or a fragment thereof. Where the intron is notnaturally occurring within the gene (as for most cDNAs), the intron(s)may be obtained from another source. The position of the intron withrespect to flanking sequences and the Fhm gene is generally important,as the intron must be transcribed to be effective. Thus, when an FhmcDNA molecule is being transcribed, the preferred position for theintron is 3′ to the transcription start site, and 5′ to the polyAtranscription termination sequence. Preferably, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt the this coding sequence. Any intronfrom any source, including viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this invention, provided thatit is compatible with the host cell(s) into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

The expression and cloning vectors of the present invention will eachtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the Fhm polypeptide.

Promoters are untranscribed sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of a particular molecule, suchas that encoding Fhm. Promoters are conventionally grouped into one oftwo classes, inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the othere hand, initiate continuous geneproduction; that is, there is little or no control over gene expression.A large number of promoters, recognized by a variety of potential hostcells, are well known. A suitable promoter is operably linked to the DNAencoding Fhm by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence into thevector. The native Fhm promoter sequence may be used to directamplification and/or expression of Fhm encoding nucleic acid molecule. Aheterologous promoter is preferred, however, if it permits greatertranscription and higher yields of the expressed protein as compared tothe native promoter, and if it is compatible with the host cell systemthat has been selected for use.

Promoters suitable for use with prokaryotic hosts include, but are notlimited to the beta-lactamase and lactose promoter systems; alkalinephosphatase, a tryptophan (trp) promoter system; and hybrid promoterssuch as the tac promoter. Other known bacterial promoteres and alsosuitable. Their sequences have been published, thereby enabling oneskilled in the art to ligate them to the desired DNA sequence(s), usinglinkers or adapters as needed to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowl pox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retrovirus, hepatitis-B virus, herpes virus and mostpreferably Simian Virus 40 (SV40). Other suitable mammalian promotersinclude heterologous mammalian promoters, e.g., heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest in controlling Fhmtranscription include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature, 290:304-310, 1981); the CMVpromoter; the promoter contained in the 3′ long terminal repeat (LTR) ofRous sarcoma virus (RSV) (Yamamoto, et al., Cell, 22:787-797, 1980); theherpes thymidine kinase (TK) promoter (Wagner et al., Proc. Natl. Acad.Sci. U.S.A., 78:144-1445, 1981); the regulatory sequences of themetallothionine gene (Brinster et al., Nature, 296:39-42, 1982);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731,1978); or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci.U.S.A., 80:21-25, 1983). Also of use are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al., Cell,38:639-646, 1984; Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409, 1986; MacDonald, Hepatology, 7:425-515, 1987); the insulingene control region which is active in pancreatic beta cells (Hanahan,Nature, 315:115-122, 1985); the immunoglobulin gene control region whichis active in lymphoid cells (Grosschedl et al., Cell, 38:647-658, 1984;Adames et al., Nature, 318:533-538, 1985; Alexander et al, Mol. Cell.Biol., 7:1436-1444, 1987); the mouse mammary tumor virus control regionwhich is active in testicular, breast, lymphoid and mast cells (Leder etal., Cell, 45:485-495, 1986), the albumin gene control region which isactive in liver (Pinkert et al., Genes and Devel., 1:268-276, 1987); thealphafetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol., 5:1639-1648, 1985; Hammer et al., Science,235:53-58, 1987); the alpha 1-antitrypsin gene control region which isactive in the liver (Kelsey et al., Genes and Devel., 1:161-171, 1987);the beta-globin gene control region which is active in myeloid cells(Mogram et al., Nature, 315:338-340, 1985; Kollias et al., Cell,46:89-94, 1986); the myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., Cell,48:703-712, 1987); the myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, Nature, 314:283-286, 1985); and thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science, 234:1372-1378, 1986).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding a Fhm polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease its transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation or upregulation of eukaryotic promoters. While anenhancer may be spliced into the vector at a position 5′ or 3′ to Fhmnucleic acid molecules, it is typically located at a site 5′ from thepromoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe desired flanking sequences set forth above are not already presentin the vector, they may be individually obtained and ligated into thevector. Methods used for obtaining each of the flanking sequences arewell known to one skilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, Carlsbad, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), pDSR-alpha (PCT Publ. No. WO 90/14364) andpFastBacDual (Gibco-Brl Grand Island, N.Y.).

Additional suitable vectors include, but are not limited to, cosmids,plasmids or modified viruses, but it will be appreciated that the vectorsystem must be compatible with the selected host cell. Such vectorsinclude, but are not limited to plasmids such as Bluescript® plasmidderivatives (a high copy number ColE1-based phagemid, Stratagene CloningSystems Inc., La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeastor virus vectors such as a baculovirus expression system (pBacPAKplasmid derivatives, Clontech, Palo Alto, Calif.).

After the vector has been constructed and a nucleic acid moleculeencoding an Fhm polypeptide has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an Fhm polypeptide into a selected host cellmay be accomplished by well-known methods such as transfection,infection, calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,supra.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (yeast, insect, or vertebrate cells). The host cell, whencultured under appropriate conditions, synthesizes an Fhm polypeptidewhich can subsequently be collected from the culture medium (if the hostcell secretes it into the medium) or directly from the host cellproducing it (if it is not secreted). The selection of an appropriatehost cell will depend upon various factors, such as desired expressionlevels, polypeptide modifications that desirable or necessary foractivity, (such as glycosylation or phosphorylation), and ease offolding into a biologically active molecule.

Yeast and mammalian cells are preferred hosts of the present invention.The use of such hosts provides substantial advantages in that they canalso carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in these hosts.

Yeast recognize leader sequences on cloned mammalian gene products andsecrete peptides bearing leader sequences (i.e., pre-peptides).Mammalian cells provide post-translational modifications to proteinmolecules including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblastorigin such as VERO or CHO-K1, and their derivatives. For a mammalianhost, several possible vector systems are available for the expressionof the desired Fhm protein. A wide variety of transcriptional andtranslational regulatory sequences may be employed, depending upon thenature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Usefulsignals are regulatory signals which are temperature-sensitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired receptor molecule does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the desired receptor molecule encoding DNA sequence) ora frame-shift mutation (if the AUG codon is not in the same readingframe as the desired Fhm protein encoding sequence).

The expression of the Fhm proteins can also be accomplished inprocaryotic cells. Preferred prokaryotic hosts include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F⁻, lambda⁻, prototrophic (ATCC27325)), and other enterobacteria (such as Salmonella typhimurium orSerratia marcescens), and various Pseudomonas species. The prokaryotichost must be compatible with the replicon and control sequences in theexpression plasmid.

To express the desired Fhm protein in a prokaryotic cell (such as, forexample, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it isnecessary to operably link the desired receptor molecule encodingsequence to a functional prokaryotic promoter. Such promoters may beeither constitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, and the bla promoter of the β-lactamasegene of pBR322, etc. Examples of inducible prokaryotic promoters includethe major right and left promoters, of bacteriophage λ (P_(L) andP_(R)), the trp, recA, lacZ, lacI, gal, and tac promoters of E. coli,the α-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985), theσ-28-specific promoters of B. subtilis (Gilma et al., Gene 32:11-20,1984), the promoters of the bacteriophages of Bacillus (Gryczan, T. J.,In: The Molecular Biology of the Bacilli, Academic Press, Inc., NewYork, 1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet.203:468-478 1986). Prokaryotic promoters are reviewed by Glick, (J. Ind.Microbiol. 1:277-282, 1987); Cenatiempo, Biochimie 68:505-516, 1986);and Gottesman, Ann. Rev. Genet. 18:415-442 1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream from the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold et al. (Ann.Rev. Microbiol. 35:365-404, 1981).

The desired Fhm protein encoding sequence and an operably linkedpromoter may be introduced into a recipient prokaryotic or eukaryoticcell either as a non-replicating DNA (or RNA) molecule, which may eitherbe linear or, more preferably, a closed covalent circular molecule.Since such molecules are incapable of autonomous replication, theexpression of the desired receptor molecule may occur through thetransient expression of the introduced sequence. Alternatively,permanent expression may occur through the integration of the introducedsequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay complement an auxotrophy in the host (such as leu21, or ura3, whichare common yeast auxotrophic markers), biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene can either be directly linked to the DNA genesequences to be expressed, or introduced into the same cell byco-transfection.

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include, for e.g. the ease with which recipient cells thatcontain the vector may be recognized and selected from those recipientcells which do not contain the vector; the number of copies of thevector which are desired in a particular host; and whether it isdesirable to be able to “shuttle” the vector between host cells ofdifferent species.

Any of a series of yeast gene expression systems can also be utilized.Examples of such expression vectors include the yeast-2-micron circle,the expression plasmids YEP13, YVP and YRP, etc., or their derivatives.Such plasmids are well known in the art (Botstein, et al., Miami Wntr.Symp. 19:265-274 (1982); Broach, In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, Cell28:203-204 1982).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper or the like. The selectablemarker gene can either be directly linked to the DNA sequences to beexpressed, or introduced into the same cell by co-transformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,Mol. Cell. Biol. 3:280 1983, and others. Preferred eukaryotic vectorsinclude PWLNEO, PSV2CAT, POG44, PXT1, pSG, pSVK3, pBPV, pMSG, pSVL(Pharmacia).

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli such as, for example, pBR322, ColE1, pSC101,pACYC 184, πVX, pQE70, pQE60, pQE9, pBG, pD10, Phage script, psix174,pbmescript SK, pbsks, pNH8A, pNHIBa, pNH18A, pNH46A (SL rare gone),ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Such plasmids are, forexample, disclosed by Maniatis, T., et al. (In: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Suchplasmids are disclosed by Gryczan, T. (In: The Molecular Biology of theBacilli, Academic Press, New York (1982), pp. 307-329). SuitableStreptomyces plasmids include pISJ101 (Kendall, et al., J. Bacteriol.169:4177-4183 1987), and Streptomyces bacteriophages such as φC31(Chater, et al., In: Sixth International Symposium on ActinomycetalesBiology, Akademiai Kaido, Budapest, Hungary, 1986, pp 45-541).Pseudomonas plasmids are reviewed by John, et al. (Rev. Infect. Dis.8:693-704, 1986, and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 1978).However, any other plasmid or vector may be used as long as they arereplicable and viable in the host cell.

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced into anappropriate host. Various techniques may be employed, such as aprotoplast fusion, calcium phosphate precipitation, electroporation orother conventional techniques. After the fusion, the cells are grown inmedia and screened for appropriate activities. Expression of thesequence results in the production of the Fhm protein.

Suitable host cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO; ATCC No. CCL61), CHO DHFR cells(Urlaub et al. Proc. Natl. Acad. Sci. U.S.A, 97: 4216-4220, 1980) humanembryonic kidney (HEK), 293 or 293T cells (ATCC No. CRL 1573), or 3T3cells (ATCC No. CRL920). The selection of suitable mammalian host cellsand methods for transformation, culture, amplification, screening,product production and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 (ATCC No. CRL 1650) and COS-7(ATCC No. CRL 1651) cell lines, and the CV-1 (ATCC No. CCL70) cell line.Further exemplary mammalian host cells include primate cell lines androdent cell lines, including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain a dominantacting selection gene. Other suitable mammalian cell lines include, butare not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaKhamster cell lines, which are available from the ATCC. Each of thesecell lines is known by and available to those skilled in the art ofprotein expression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α (ATCC No. 33694), DH10, and MC1061 (ATCC No. 53330)) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomycesspp., and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Preferred yeast strains include, for example,Saccharomyces cerevisiae and Pichia pastoris.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniquies, 14:810-817, 1993), Lucklow (Curr. Opin.Biotechnol., 4:564-572, 1993) and Lucklow et al. (J. Virol.,67:4566-4579, 1993). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

One may also use transgenic animals to express glycosylated Fhmpolypeptides. For example, one may use a transgenic milk-producinganimal (e.g. a cow or goat) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce Fhmpolypeptides; however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

Polypeptide Production

Host cells comprising an Fhm polypeptide expression vector (i.e.,transformed or transfected) may be cultured using standard media wellknown to the skilled artisan. The media will usually contain allnutrients necessary for the growth and survival of the cells. Suitablemedia for culturing E. coli cells include for example, Luria Broth (LB)and/or Terrific Broth (TB). Suitable media for culturing eukaryoticcells are Rosewell Park Memorial Media 1640 (RPMI 1640), MinimalEssential Media (MEM), Dulbecco's Modified Eagles Media (DMEM), all ofwhich may be supplemented with serum and/or growth factors as indicatedby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transformed cells is added as a supplement to the media. The compoundto be used will be dictated by the selectable marker element present onthe plasmid with which the host cell was transformed. For example, wherethe selectable marker element is kanamycin resistance, the compoundadded to the culture medium will be kanamycin. Other compunds forselctive growth media include ampicillin, tetracycline and neomycin. Theamount of Fhm polypeptide produced by a host cell can be evaluated usingstandard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If a Fhm polypeptide has been designed to be secreted from the hostcells, the majority of polypeptide may be found in the cell culturemedium. If however, the Fhm polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or nucleus (foreukaryotic host cells) or in the cytosol (bacterial host cells).

The intracellular material (including inclusion bodies for gram-negativebacteria) can be extracted from the host cell using any standardtechnique known to the skilled artisan. For example, the host cells canbe lysed to release the contents of the periplasm/cytoplasm by Frenchpress, homogenization, and/or sonication followed by centrifugation.

If a Fhm polypeptide has formed inclusion bodies in the cytosol, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with a chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The Fhm polypeptide in its now soluble form can thenbe analyzed using gel electrophoresis, immunoprecipitation or the like.If it is desired to isolate the Fhm polypeptide, isolation may beaccomplished using standard methods such as those described herein andin Marston et al. (Meth. Enz., 182:264-275 1990).

In some cases, a Fhm polypeptide may not be biologically active uponisolation. Various methods for “refolding” or converting the polypeptideto its tertiary structure and generating disulfide linkages, can be usedto restore biological activity. Such methods include exposing thesolubilized polypeptide to a pH usually above 7 and in the presence of aparticular concentration of a chaotrope. The selection of chaotrope isvery similar to the choices used for inclusion body solubilization, butusually the chaotrope is used at a lower concentration and is notnecessarily the same as chaotropes used for the solubilization. In mostcases the refolding/oxidation solution will also contain a reducingagent or the reducing agent plus its oxidized form in a specific ratioto generate a particular redox potential allowing for disulfideshuffling to occur in the formation of the protein's cysteine bridge(s).Some of the commonly used redox couples include cysteine/cystamine,glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, 2-mercaptoethanol(βME)/dithio-β(ME). Acosolvent is necessary to increase the efficiency of the refolding, andthe more common reagents used for this purpose include glycerol,polyethylene glycol of various molecular weights, arginine and the like.

If inclusion bodies are not formed to a significant degree uponexpression of a Fhm polypeptide, then the polypeptide will be foundprimarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide and may be further isolated from thesupernatant using methods such as those described herein.

The purification of an Fhm polypeptide from solution can be accomplishedusing a variety of techniques. If the polypeptide has been synthesizedsuch that it contains a tag such as Hexahistidine (Fhmpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at eitherits carboxyl or amino terminus, it may be purified in a one-step processby passing the solution through an affinity column where the columnmatrix has a high affinity for the tag.

For example, polyhistidine binds with great affinity and specificity tonickel, thus an nickel; thus affinity column of nickel (such as theQiagen® nickel columns) can be used for purification of Fhmpolypeptide/polyHis. See for example, Ausubel et al., eds., CurrentProtocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, NewYork 1993.

Additionally, the Fhm polypeptide may be purified through the use of amonoclonal antibody which is capable of specifically recognizing andbinding to the Fhm polypeptide.

Suitable procedures for purification thus include, without limitation,affinity chromatography, immunoaffinity chromatography, ion exchangechromatography, molecular sieve chromatography, High Performance LiquidChromatography (HPLC), electrophoresis (including native gelelectrophoresis) followed by gel elution, and preparative isoelectricfocusing (“Isoprime” machine/technique, Hoefer Scientific, SanFrancisco, Calif.). In some cases, two or more purification techniquesmay be combined to achieve increased purity.

Fhm polypeptides, fragments, and/or derivatives thereof may also beprepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art, such as those set forth byMerrifield et al., (J. Am. Chem. Soc., 85:2149, 1963), Houghten et al.(Proc Natl Acad. Sci. USA, 82:5132, 1985), and Stewart and Young (SolidPhase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984).Such polypeptides may be synthesized with or without a methionine on theamino terminus. Chemically synthesized Fhm polypeptides or fragments maybe oxidized using methods set forth in these references to formdisulfide bridges. Chemically synthesized Fhm polypeptides, fragments orderivatives are expected to have comparable biological activity to thecorresponding Fhm polypeptides, fragments or derivatives producedrecombinantly or purified from natural sources, and thus may be usedinterchangeably with recombinant or natural Fhm polypeptide.

Another means of obtaining Fhm polypeptide is via purification frombiological samples such as source tissues and/or fluids in which the Fhmpolypeptide is naturally found. Such purification can be conducted usingmethods for protein purification as described above. The presence of theFhm polypeptide during purification may be monitored, for example, usingan antibody prepared against recombinantly produced Fhm polypeptide orpeptide fragments thereof.

A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and the methods can be used toproduce polypeptides having specificity for Fhm. See for example,Roberts et al., Proc. Natl. Acad. Sci. USA, 94:12297-12303, 1997, whichdescribes the production of fusion proteins between an mRNA and itsencoded peptide. See also Roberts, Curr. Opin. Chem. Biol., 3:268-273,1999.

Additionally, U.S. Pat. No. 5,824,469 describes methods of obtainingoligonucleotides capable of carrying out a specific biological function.The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those which exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated. U.S. Pat. Nos.5,763,192, 5,814,476, 5,723,323, and 5,817,483 describe processes forproducing peptides or polypeptides. This is done by producing stochasticgenes or fragments thereof, and then introducing these genes into hostcells which produce one or more proteins encoded by the stochasticgenes. The host cells are then screened to identify those clonesproducing peptides or polypeptides having the desired activity.

Another method for producing peptides or polypeptides is described inPCT/US98/20094 (WO99/15650) filed by Athersys. Inc. Known as “RandomActivation of Gene Expression for Gene Discovery” (RAGE-GD), the processinvolves the activation of endogenous gene expression or over-expressionof a gene by in situ recombination methods. For example, expression ofan endogenous gene is activated or increased by integrating a regulatorysequence into the target cell which is capable of activating expressionof the gene by non-homologous or illegitimate recombination. The targetDNA is first subjected to radiation, and a genetic promoter inserted.The promoter eventually locates a break at the front of a gene,initiating transcription of the gene. This results in expression of thedesired peptide or polypeptide.

It will be appreciated that these methods can also be used to createcomprehensive IL-17 like protein expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

Proteins, Polypeptides, Fragments, Variants and Muteins of Fhm:

Polypeptides of the invention include isolated Fhm polypeptides andpolypeptides related thereto including fragments, variants, fusionpolypeptides, and derivatives as defined hereinabove.

Fhm fragments of the invention may result from truncations at the aminoterminus (with or without a leader sequence), truncations at the carboxyterminus, and/or deletions internal to the polypeptide. Most deletionsand insertions, and substitutions in particular, are not expected toproduce radical changes in the characteristics of the Fhm protein.However, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, one skilledin the art will appreciate that the effect will be evaluated by routinescreening assays. For example, a variant typically is made bysite-specific mutagenesis of the Fhm-encoding nucleic acid, expressionof the variant nucleic acid in recombinant cell culture, and,optionally, purification from the cell culture, for example, byimmunoaffinity adsorption on a polyclonal anti-Fhm antibody column (toabsorb the variant by binding it to at least one remaining immuneepitope). In preferred embodiments, truncations and/or deletionscomprise about 10 amino acids, or about 20 amino acids, or about 50amino acids, or about 75 amino acids, or about 100 amino acids, or morethan about 100 amino acids. The polypeptide fragments so produced willcomprise about 25 contiguous amino acids, or about 50 amino acids, orabout 75 amino acids, or about 100 amino acids, or about 150 aminoacids, or about 200 amino acids. Such Fhm polypeptides fragments mayoptionally comprise an amino terminal methionine residue.

Fhm polypeptide variants of the invention include one or more amino acidsubstitutions, additions and/or deletions as compared to SEQ ID NO: 4.In preferred embodiments, the variants have from 1 to 3, or from 1 to 5,or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, orfrom 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 aminoacid substitutions, insertions, additions and/or deletions, wherein thesubstitutions may be conservative, as defined above, or non-conservativeor any combination thereof. More particularly, Fhm variants may comprisethe amino acid sequence set out as SEQ ID NO: 4, wherein one or moreamino acids from the group consisting of amino acids 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176 up to 251 is substituted withanother amino acid. The variants may have additions of amino acidresidues either at the carboxy terminus or at the amino terminus (withor without a leader sequence).

Preferred Fhm polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites has been alteredcompared to native Fhm polypeptide. In one embodiment, Fhm variantscomprise a greater or a lesser number of N-linked glycosylation sites. AN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Thr, where the amino acid residue designated as X may be any type ofamino acid except proline. Substitution(s) of amino acid residues tocreate this sequence provides a potential new site for addition of aN-linked carbohydrate chain. Alternatively, substitutions to eliminatethis sequence will remove an existing N-linked carbohydrate chain. Alsoprovided is a rearrangement of N-linked carbohydrate chains wherein oneor more N-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated.

One skilled in the art will be able to determine suitable variants ofthe native Fhm polypeptide using well known techniques. For example, onemay be able to predict suitable areas of the molecule that may bechanged without destroying biological activity. Also, one skilled in theart will realize that even areas that may be important for biologicalactivity or for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

For predicting suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of Fhm polypeptide to such similar polypeptides. After makingsuch a comparison, one skilled in the art would be able to determineresidues and portions of the molecules that are conserved among similarpolypeptides. One skilled in the art would know that changes in areas ofthe Fhm molecule that are not conserved would be less likely toadversely affect biological activity and/or structure. One skilled inthe art would also know that, even in relatively conserved regions, onecould have likely substituted chemically similar amino acids for thenaturally occurring residues while retaining activity (e.g. conservativeamino acid residue substitutions).

Also, one skilled in the art may review structure-function studiesidentifying residues in similar polypeptides that are important foractivity or structure. In view of such a comparison, one skilled in theart can predict the importance of amino acid residues in Fhm thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of Fhm.

If available, one skilled in the art can also analyze the crystalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of that information, one skilled in theart may be able to predict the alignment of amino acid residues of Fhmpolypeptide with respect to its three dimensional structure. One skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules.

Moreover, one skilled in the art can generate test variants containing asingle amino acid substitution at each amino acid residue. The variantscan be screened using activity assays disclosed in this application.Such variants are used to gather information about suitable variants.For example, if one discovered that a change to a particular amino acidresidue resulted in destroyed activity, variants with such a changewould be avoided. Thus, based on information gathered from suchexperiments, when attempting to find additional acceptable variants, oneskilled in the art can determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

Fhm polypeptide analogs of the invention can be determined by comparingthe amino acid sequence of Fhm polypeptide with related family members.Exemplary Fhm polypeptide related family members include, but are notlimited to, the TNF-α, TNK-β, LyT-β, FasL, CD40L, CD30L, OPGL, andTRAIL. This comparison can be accomplished by using a Pileup alignment(Wisconsin GCG Program Package) or an equivalent (overlapping)comparison with multiple family members within conserved andnon-conserved regions.

As shown in FIG. 1, the predicted amino acid sequence of Fhm polypeptide(SEQ ID NO: 4) is aligned with the corresponding regions of human FasL,mouse FasL, rat FasL, human CD40L, mouse CD40L, mouse OPGL, human OPGL,human TRAIL, mouse TRAIL, human CD30L, human CD30L, human LyT-β, mouseLyT-β, human TNF-β, mouse TNF-β, human TNF-α and mouse TNF-α. (SEQ IDNOS: 5-21). Other Fhm polypeptide analogs can be determined using theseor other methods known to those of skill in the art. These overlappingsequences provide guidance for conservative and non-conservative aminoacids substitutions resulting in additional Fhm analogs. It will beappreciated that these amino acid substitutions can consist of naturallyoccurring or non-naturally occurring amino acids. For example, asdepicted in FIG. 1, alignment of the B/B′ loop and D/E loop of theseligands indicates potential Fhm analogs may have the Val residue atposition 153 substituted with a lie, Met, Leu, Phe, Ala or Norleucineresidue, the Tyr residue at position 147 may be substituted with, or thePhe residue at position 154 may be substituted with Leu, Val, Ile, Ala,or Tyr residue. Further, the Ser residue at position 151 may besubstituted with Thr, Ala, or Cys, the Gly residue at 145 may besubstituted with Pro or Ala, and the Tyr at position 150 may besubstituted with Trp, Phe, Thr or Ser.

Fhm fusion polypeptides of the invention comprise Fhm polypeptides,fragments, variants, or derivatives fused to one or more heterologouspeptides or proteins. Heterologous peptides and proteins include, butare not limited to, an epitope to allow for detection and/or isolationof a Fhm fusion polypeptide, a transmembrane receptor protein or aportion thereof, such as an extracellular domain, or a transmembrane, aligand or a portion thereof which binds to a transmembrane receptorprotein, an enzyme or portion thereof which is catalytically active, aprotein or peptide which promotes oligomerization, such as leucinezipper domain, and a protein or peptide which increase stability, suchas an immunoglobulin constant region. A Fhm polypeptide may be fused toitself or to a fragment, variant, or derivative thereof. Fusions may bemade either at the amino terminus or at the carboxy terminus of a Fhmpolypeptide, and may be direct with no linker or adapter molecule or maybe through a linker or adapter molecule, such as one or more amino acidresidues up to about 20 amino acids residues, or up to about 50 aminoacid residues. Alternatively, the Fhm fusion-protein may comprise one ortwo Fhm polypeptides covalently linked to one or two TNF ligandpolypeptide(s), or a member of the TNF ligand family or a cytokinereceptor such as interleukin-1 (IL-1) polypeptide. The ligandspreferably are produced as fusion proteins using recombinant DNAtechnology. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for proteolyticcleavage to allow for separation and subsequent folding of the fusedmoieties.

Also envisioned as a part of the invention are circularly permutedstructural analogs of the Fhm polypeptide.

The development of recombinant DNA methods has made it possible to studythe effects of sequence transposition on protein folding, structure andfunction. The approach used in creating new sequences resembles that ofnaturally occurring pairs of proteins that are related by linearreorganization of their amino acid sequences (Cunningham, et al., Proc.Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979; Teather & Erfle, J.Bacteriol. 172:3837-3841, 1990; Schimming et al., Eur. J. Biochem.204:13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett 260:127-130, 1991;MacGregor et al., FEBS Lett. 378:263-266, 1996). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). A newN-terminus is selected at an internal site (breakpoint) of the originalsequence, the new sequence having the same order of amino acids as theoriginal from the breakpoint until it reaches an amino acid that is ator near the original C-terminus. At this point the new sequence isjoined, either directly or through an additional portion of sequence(linker), to an amino acid that is at or near the original N-terminus,and the new sequence continues with the same sequence as the originaluntil it reaches a point that is at or near the amino acid that wasN-terminal to the breakpoint site of the original sequence, this residueforming the new C-terminus of the chain.

This approach has been applied to proteins which range in size from 58to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol.165:407-413,1983; Li & Coffino, Mol. Cell. Biol. 13:2377-2383, 1993).The proteins examined have represented a broad range of structuralclasses, including proteins that contain predominantly α-helix(interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995),predominantly β-sheet (interleukin-1; Horlick et al., Protein Eng.5:427-431, 1992), or mixtures of the two (yeast phosphoribosylanthranilate isomerase; Luger et al., Science 243:206-210, 1989).

In a preferred embodiment, a Fhm polypeptide, fragment, variant and/orderivative is fused to an Fc region of human IgG. In one example, ahuman IgG hinge, CH2 and CH3 region may be fused at either theN-terminus or C-terminus of the Fhm polypeptides using methods known tothe skilled artisan. In another example, a portion of a hinge regionsand CH2 and CH3 regions may be fuse. The Fhm Fc-fusion polypeptide soproduced may be purified by use of a Protein A affinity column (Pierce,Rockford, Ill.). In addition, peptide and proteins fused to an Fc regionhave been found to exhibit a substantially greater half-life in vivothan the unfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be naturally occurring Fc region, or may be altered to improvecertain qualities such as therapeutic qualities, circulation time,reduce aggregation, etc.

Fhm polypeptide derivatives are also included in the scope of thepresent invention. Covalent modifications of the Fhm proteins of thepresent invention are included within the scope of this invention.Variant Fhm proteins may be conveniently prepared by in vitro synthesis.Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the purified or crude protein with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The resulting covalent derivatives areuseful in programs directed at identifying residues important forbiological activity.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carbocyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic orcarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineEpsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commoly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R¹) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking theFhm protein(s)/polypeptide to water-insoluble support matrixes orsurfaces for use in the method for cleaving the Fhm protein-fusionpolypeptide to release and recover the cleaved polypeptide. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctional imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimides such as bix-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[p-azidophenyl) dithio]propioimidate yield photoactivatableintermediates that are capable of forming cross links in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440, incorporated herein by reference, are employed forprotein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86,1983),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups. Such derivatives are chemicallymodified Fhm polypeptide compositions in which Fhm polypeptide is linkedto a polymer. The polymer selected is typically water soluble so thatthe protein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer selectedis usually modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled as provided for in the present methods.The polymer may be of any molecular weight, and may be branched orunbranched. Included within the scope of the Fhm polypeptide polymers isa mixture of polymers. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 k kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is between about 5 kDa and 5 kDa, about50 kDa, more preferably between about 12 kDa to about 40 kDa and mostpreferably between about 20 kDa to about 35 kDa.

Suitable water soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,carbohydrates; sugars; phosphates; polyethylene glycol (PEG) (includingthe forms of PEG that have been used to derivatize proteins, includingmono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol);monomethoxy-polyethylene glycol; dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose; cellulose; othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol. Also encompassed by the present invention arebifunctional crosslinking molecules which may be used to preparecovalently attached multimers of the polypeptide comprising the aminoacid sequence of SEQ ID NO: 4 or an Fhm polypeptide variant.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of SEQ If) NO: 4, or anFhm polypeptide variant becomes attached to one or more polymermolecules, and (b) obtaining the reaction product(s). The optimalreaction conditions will be determined based on known parameters and thedesired result. For example, the larger the ratio of polymermolecules:protein, the greater the percentage of attached polymermolecule. In one embodiment, the Fhm polypeptide derivative may have asingle polymer molecule moiety at the amino terminus. (See, e.g., U.S.Pat. No. 5,234,784).

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol. PEG is a linear or branched neutralpolyether, available in a broad range of molecular weights, and issoluble in water and most organic solvants. PEG is effective atexcluding other polymers or peptides when present in water, primarilythrough its high dynamic chain mobility and hydrophibic nature, thuscreating a water shell or hydration sphere when attached to otherproteins or polymer surfaces. PEG is nontoxic, non-immunogenic, andapproved by the Food and Drug Administration for internal consumption.

Proteins or enzymes when conjugated to PEG have demonstratedbioactivity, non-antigenic properties, and decreased clearance rateswhen administered in animals. F. M. Veronese et al., Preparation andProperties of Moonomthoxypoly(ethylene glyco.)-modified Enzymes forTherapeutic Applications, in J. M. Harris ed., Poly(Ethylene Clycol)Chemistry—Biotechnical and Biomedical Applications 127-36, 1992,incorporated herein by reference. This is due to the exclusionproperties of PEG in preventing recognition by the immune system. Inaddition, PEG has been widely used in surface modification procedures todecrease protein adsorption and improve blood compatibility. S. W. Kimet al, Ann. N. Y Acad. Sci. 516: 116-30 1987; Jacobs et al., Artif.Organs 12: 500-501, 1988; Park et al., J. Poly. Sci, Part A 29:1725-31,1991, incorporated herein by reference. Hydrophobic polymer surfaces,such as polyurethanes and polystyrene were modified by the grafting ofPEG (MW 3,400) and employed as nonthrombogenic surfaces. In thesestudies, surface properties (contact angle) were more consistent withhydrophilic surfaces, due to the hydrating effect of PEG. Moreimportantly, protein (albumin and other plasma proteins) adsorption wasgreatly reduced, resulting from the high chain motility, hydrationsphere, and protein exclusion properties of PEG.

PEG (MW 3,4000) was determined as an optimal size in surfaceimmobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45,1992, while PEG (MW 5,000) was most beneficial in decreasing proteinantigenicity. (F. M. Veronese et al., In J. M. Harris et., Poly(EthyleneGlycol) Chemistry—Biotechnical and Biomedical Applications 127-36,supra., incorporated herein by reference)

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated Fhmpolypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby Fhm polypeptidebecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG: protein, thegreater the percentage of poly-pegylated product.

In a preferred embodiment, the Fhm polypeptide derivative will have asingle PEG moiety at the N terminus. See U.S. Pat. No. 8,234,784, hereinincorporated by reference.

Generally, conditions which may be alleviated or modulated byadministration of the present Fhm polypeptide derivative include thosedescribed herein for Fhm polypeptides. However, the Fhm polypeptidederivative disclosed herein may have additional activities, enhanced orreduced biological activity, or other characteristics, such as increasedor decreased half-life, as compared to the non-derivatized molecules.

Genetically Engineered Non-Human Animals

Additionally included within the scope of the present invention arenon-human animals such as mice, rats, or other rodents, rabbits, goats,or sheep, or other farm animals, in which the gene (or genes) encodingthe native Fhm polypeptide has (have) been disrupted (“knocked out”)such that the level of expression of this gene or genes is(are)significantly decreased or completely abolished. Such animals may beprepared using techniques and methods such as those described in U.S.Pat. No. 5,557,032.

The present invention further includes non-human animals such as mice,rats, or other rodents, rabbits, goats, sheep, or other farm animals, inwhich either the native form of the Fhm gene(s) for that animal or aheterologous Fhm gene(s) is (are) over-expressed by the animal, therebycreating a “transgenic” animal. Such transgenic animals may be preparedusing well known well-known methods such as those described in U.S. Pat.No. 5,489,743 and PCT application No. WO94/28122, Application No. WO94/28122.

The present invention further includes non-human animals in which thepromoter for one or more of the Fhm polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods) to alter the level of expression of one or moreof the native Fhm polypeptides.

These non-human animals may be used for drug candidate screening. Insuch screening, the impact of a drug candidate on the animal may bemeasured; for example, drug candidates may decrease or increase theexpression of the Fhm gene. In certain embodiments, the amount of Fhmpolypeptide, that is produced may be measured after the exposure of theanimal to the drug candidate. Additionally, in certain embodiments, onemay detect the actual impact of the drug candidate on the animal. Forexample, the overexpression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

Microarray

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array has numerous copies of asingle species of DNA which acts as a target for hybridization for itscognate mRNA. In expression profiling using DNA microarray technology,mRNA is first extracted from a cell or tissue sample and then convertedenzymatically to fluorescently labeled cDNA. This material is hybridizedto the microarray and unbound cDNA is removed by washing. The expressionof discrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA which is specifically bound toeach target DNA. In this way, the expression of thousands of genes canbe quantitated in a high throughput, parallel manner from a singlesample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the Fhm molecules of the invention,including, but not limited to: the identification and validation of Fhmdisease-related genes as targets for therapeutics; molecular toxicologyof Fhm molecules and inhibitors thereof; stratification of populationsand generation of surrogate markers for clinical trials; and enhancingFhm-related small molecule drug discovery by aiding in theidentification of selective compounds in high throughput screens (HTS).

Selective Binding Agents

As used herein, the term “selective binding agent” refers to a moleculewhich has specificity for one or more Fhm polypeptides. Suitableselective binding agents include, but are not limited to, antibodies andderivatives thereof, polypeptides, and small molecules. Suitableselective binding agents may be prepared using methods known in the art.An exemplary Fhm polypeptide selective binding agent of the presentinvention is capable of binding a certain portion of the Fhm polypeptidethereby inhibiting the binding of the polypeptide to the Fhm polypeptidereceptor(s).

Selective binding agents such as antibodies and antibody fragments thatbind Fhm polypeptides are within the scope of the present invention. Theantibodies may be polyclonal including monospecific polyclonal,monoclonal (mAbs), recombinant, chimeric, humanized such as CDR-grafted,human, single chain, and/or bispecific, as well as fragments, variantsor derivatives thereof Antibody fragments include those portions of theantibody which bind to an epitope on the Fhm polypeptide. Examples ofsuch fragments include Fab and F(ab′) fragments generated by enzymaticcleavage of full-length antibodies. Other binding fragments includethose generated by recombinant DNA techniques, such as the expression ofrecombinant plasmids containing nucleic acid sequences encoding antibodyvariable regions.

Polyclonal antibodies directed toward a Fhm polypeptide generally areproduce in animals (e.g. rabbits or mice) by means of multiplesubcutaneous or intraperitoneal injections of Fhm and an adjuvant. Itmay be useful to conjugate a Fhm polypeptide, or a variant, fragment orderivative thereof to a carrier protein that is immunogenic in thespecies to be immunized, such as keyhole limpet heocyanin, serum,albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also,aggregating agents such as alum are used to enhance the immune response.After immunization, the animals are bled and the serum is assayed foranti-Fhm antibody titer.

Monoclonal antibodies directed toward Fhm are produced using any methodwhich provides for the production of antibody molecules by continuouscell lines in culture. Examples of suitable methods for preparingmonoclonal antibodies include the hybridoma method of Kohler et al.,Nature 256: 495-497, 1975, and the human B-cell hybridoma method,Kozbor, J. Immunol. 133: 3001, 1984; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

Also provided by the invention are hybridoma cell lines which producemonoclonal antibodies reactive with Fhm polypeptides.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is/are identical with or a homologous tocorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; Morrison, et al.,Proc. Natl. Acad. Sci. U.S.A. 81: 6851-6855, 1985; incorporated hereinby reference).

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art (see U.S. Pat. Nos. 5,585,089 and 5,693,762).Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. Humanization can beperformed, for example, methods described in the art (Jones et al.,Nature 321: 522-525, 1986; Riechmann et al., Nature, 332: 323-327, 1988;Verhoeyen et al., Science 239: 1534-1536, 1988), by substituting atleast a portion of a rodent complementarity-determining region (CDR) forthe corresponding regions of a human antibody.

Also encompassed by the invention are fully human antibodies which bindFhm polypeptides, fragments, variants and/or derivatives. Suchantibodies are produced by immunization with a Fhm antigen optionallyconjugated to a carrier (i.e., at least having 6 contiguous aminoacids). Using transgenic animals (e.g., mice) that are capable ofproducing a repertoire of human antibodies in the absence of endogenousimmunoglobulin production. See, for example, Jakobovits, et al., Proc.Natl. Acad. Sci. U.S.A. 90: 2551-2555, 1993; Jakobovits, et al., Nature362: 255-258, 1993; Bruggermann, et al., Year in Immuno. 7:33, 1993. Inone method, such transgenic animals are produced by incapacitating theendogenous loci encoding the heavy and light immunoglobulin chainstherein, and inserting loci encoding human heavy and light chainproteins into the genome thereof. Partially modified animals, that isthose having less than the full complement of modifications, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies with human (rather than e.g., murine) amino acidsequences, including variable regions which are immunospecific for theseantigens. See PCT Application Nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCTapplication nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP546073A1. Human antibodies may also be produced by the expression ofrecombinant DNA in host cells or by expression in hybridoma cells asdescribed herein.

In an alternative embodiment, human antibodies can be produced inphage-display libraries (Hoogenboom, et al., J. Mol. Biol. 227:381,1991; Marks, et al., J. Mol. Biol. 222:581, 1991. These processes mimicimmune selection through the display of antibody repertoires on thesurface of filamentous bacteriophage, and subsequent selection of phageby their binding to an antigen of choice. One such technique isdescribed in PCT Application No. PCT/US98/17364, which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Monoclonal (e.g., human)antibodies may be produced by the expression of recombinant DNA in hostcells or by expression in hybridoma cells as described herein.

The anti-Fhm antibodies of the invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Sola, MonoclonalAntibodies. A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987))for the detection and quantitation of Fhm polypeptides. The antibodieswill bind Fhm polypeptides with an affinity which is appropriate for theassay method being employed.

For diagnostic applications, in certain embodiments anti-Fhm antibodiestypically may be labeled with a detectable moiety. The detectable moietycan be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,β-galactosidase, or horseradish peroxidase. See Bayer, et al., Meth.Enz. 184: 138-163, 1990.

The anti-Fhm antibodies of the invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Sola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987))for detection and quantitation of Fhm polypeptides. The antibodies willbind Fhm polypeptides with an affinity which is appropriate for theassay method being employed.

The activity of the cell lysate or purified Fhm protein variant is thenscreened in a suitable screening assay for the desired characteristic.For example, a change in the binding affinity for a ligand orimmunological character of the Fhm protein, such as affinity for a givenantibody, is measured by a competitive type immunoassay. Changes inimmunomodulation activity are measured by the appropriate assay.Modifications of such protein properties as redox or thermal stabilityhydrophobicity, susceptibility to proteolytic degradation or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to the ordinarily skilled artisan. Competitivebinding assays rely on the ability of a labeled standard (e.g., a Fhmpolypeptide, or an immunologically reactive portion thereof) to competewith the test sample analyte (a Fhm polypeptide) for binding with alimited amount of antibody. The amount of a Fhm polypeptide in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies typically are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich imuno-assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte typically is bound by a first antibody which isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three-part complex. See e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme linked immunosorbant assay (ELISA), in which case thedetectable moiety is an enzyme.

The selective binding agents, including anti-Fhm antibodies, are alsouseful for in vivo imaging. An antibody labeled with a detectable moietymay be administered to an animal, preferably into the bloodstream, andthe presence and location of the labeled antibody in the host isassayed. The antibody may be labeled with any moiety that is detectablein an animal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Selective binding agents, including antibodies of the invention, may beused as therapeutics. These therapeutic antibodies are generallyagonists or antagonists, in that they either enhance or reduce,respectively, at least one of the biological activities of a Fhmpolypeptide. In one embodiment, antagonist antibodies of the inventionare antibodies or binding fragments thereof which are capable ofspecifically binding to a Fhm polypeptide, fragment, variant and/orderivative, and which are capable of inhibiting or eliminating thefunctional activity of a Fhm polypeptide in vivo or in vitro. Inpreferred embodiments, an antagonist antibody will inhibit thefunctional activity of a Fhm polypeptide at least about 50%, preferablyat least about 80%, more preferably 90%, and most preferably 100%. Inanother embodiment, the selective binding agent may be an antibody thatis capable of interacting with an Fhm binding partner (e.g., receptor)thereby inhibiting or eliminating Fhm activity in vitro or in vivo.Selective binding agents, including agonist and antagonist anti-Fhmantibodies, are identified by screening assays which are well known inthe art.

The invention also relates to a kit comprising Fhm selective bindingagents (such as antibodies) and other reagents useful for detecting Fhmpolypeptide levels in biological samples. Such reagents may include, adetectable label, blocking serum, positive and negative control samples,and detection reagents.

The Fhm polypeptides of the present invention can be used to clone Fhmreceptors, using an expression cloning strategy. Radiolabeled(¹²⁵Iodine) Fhm polypeptide or affinity/activity-tagged Fhm polypeptide(such as an Fc fusion or an alkaline phosphatase fusion) can be used inbinding assays to identify a cell type or cell line or tissue thatexpresses Fhm receptor(s). RNA isolated from such cells or tissues canbe converted to cDNA, cloned into a mammalian expression vector, andtransfected into mammalian cells (such as COS or 293 cells) to create anexpression library. A radiolabeled or tagged Fhm polypeptide can then beused as an affinity ligand to identify and isolate from this library thesubset of cells which express the Fhm receptor(s) on their surface. DNAcan then be isolated from these cells and transfected into mammaliancells to create a secondary expression library in which the fraction ofcells expressing Fhm receptor(s) is many-fold higher than in theoriginal library. This enrichment process can be repeated iterativelyuntil a single recombinant clone containing an Fhm receptor is isolated.Isolation of the Fhm receptor(s) is useful for identifying or developingnovel agonists and antagonists of the Fhm polypeptide signaling pathway.Such agonists and antagonists include soluble Fhm receptor(s), anti-Fhmreceptor antibodies, small molecules, or antisense oligonucleotides, andthey may be used for treating, preventing, or diagnosing one or moredisease or disorder, including those described herein.

Diagnostic Kits and Reagents

This invention also contemplates use of Fhm proteins, fragments thereof,peptides, binding compositions, and their fusion products in a varietyof diagnostic kits and methods for detecting the presence of receptorsand/or antibodies. Typically the kit will have a compartment containinga Fhm peptide or gene segment or a reagent which recognizes one or theother, e.g., binding reagents.

A kit for determining the binding affinity of a binding partner or atest compound to the Fhm would typically comprise a binding partner testcompound; a labeled compound, for example an antibody having knownbinding affinity for the protein; or a source of binding partner(naturally occurring or recombinant), and a means for separating boundfrom free labeled compound, such as a solid phase for immobilizing theligand or its binding partner. Once compounds are screened, those havingsuitable binding affinity to the ligand or its binding partner can beevaluated in suitable biological assays, as are well known in the art,to determine whether they act as agonists or antagonists of Fhmactivity. The availability of recombinant Fhm and/or receptorpolypeptides also provide well defined standards for calibrating suchassays or as positive control samples.

A preferred kit for determining the concentration of, for example.Fhm-ligand and/or its cognate binding partner in a sample wouldtypically comprise a labeled compound, e.g., antibody, having knownbinding affinity for the target, a source of ligand or receptor(naturally occurring or recombinant), and a means for separating thebound from free labeled compound, for example, a solid phase forimmobilizing the ligand or receptor. Compartments containing reagents,and instructions for use or disposal, will normally be provided.

Antibodies, including antigen binding fragments, specific for the ligandor receptor, or fragments are useful in diagnostic applications todetect the presence of elevated levels of ligand, receptor, and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescence, cell cultures, body fluids, and further caninvolve the detection of antigens related to the ligand or receptor inserum, or the like. Diagnostic assays may be homogeneous (without aseparation step between free reagent and antigen complex) orheterogeneous (with a separation step). Various commercial assays exist,such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassaytechnique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), andthe like. For example, unlabeled antibodies can be employed by using asecond antibody which is labeled and which recognizes the primaryantibody to a ligand or receptor or to a particular fragment thereof.Similar assays have also been extensively discussed in the literature.(See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press.)

Anti-idiotypic antibodies may have similar uses to diagnose presence ofantibodies against a ligand or receptor, as such may be diagnostic ofvarious abnormal states. For example, overproduction of a ligand orreceptor may result in production of various immunological reactionswhich may be diagnostic of abnormal physiological states, particularlyin various inflammatory or allergic conditions.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or labeled ligand or receptor isprovided. This is usually in conjunction with other additives, such asbuffers, stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments or containers for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

The aforementioned constituents of the drug screening and the diagnosticassays may be used without modification or may be modified in a varietyof ways. For example, labeling may be achieved by covalently ornon-covalently joining a moiety which directly or indirectly provides adetectable signal. In any of these assays, the ligand, test compound,receptor, or antibodies thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating bound from the freeligand, or alternatively bound from free test compound. The ligand orreceptor can be immobilized on various matrixes, perhaps with detergentsor associated lipids, followed by washing. Suitable matrixes includeplastic such as an ELISA plate, filters, and beads. Methods ofimmobilizing the ligand or receptor to a matrix include, withoutlimitation, direct adhesion to plastic, use of a capture antibody,chemical coupling, and biotin-avidin. The last step in this approach mayinvolve the precipitation of antigen/antibody complex by any of severalmethods including those utilizing, e.g., an organic solvent such aspolyethylene glycol or a salt such as ammonium sulfate. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle et al. Clin.Chem., 30:1457-1461, 1984, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,6178, incorporated hereinby reference.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Nucleic acid molecules of the invention may be used to map the locationsof the Fhm gene and related genes on chromosomes. Mapping may be done bytechniques known in the art, such as PCR amplification, in situhybridization, and FISH.

This invention is also related to the use of the Fhm gene as part of adiagnostic assay for detecting diseases or susceptibility to diseasesrelated to the presence of mutated Fhm gene. Such diseases are relatedto an abnormal expression of Fhm, for example, abnormal cellularproliferation such as tumors and cancers.

Individuals carrying mutations in the human Fhm gene may be detected atthe DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature, 324:163-166, 1986) prior to analysis. RNA or cDNAmay also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding Fhm polypeptide can be usedto identify and analyze Fhm mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled Fhm RNA or alternativelyradiolabeled Fhm antisense DNA sequences. Perfectly matched sequencescan be distinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturing,formamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242, 1985).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85:4397-4401, 1985).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of Fhm protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, tumors, cerebral malaria and hereditary periodicfever syndromes. Assays used to detect levels of Fhm protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis, ELISA assays and “sandwich” assay. An ELISA assay (Coligan, etal., Current Protocols in Immunology, 1(2), Chapter 6, 1991) partiallycomprises preparing an antibody specific to the Fhm antigen, preferablya monoclonal antibody. In addition a reporter antibody is preparedagainst the monoclonal antibody. To the reporter antibody is attached adetectable reagent such as radioactivity, fluorescence or in thisexample a horseradish peroxidase enzyme. A sample is now removed from ahost and incubated on a solid support, e.g., a polystyrene dish, thatbinds the proteins in the sample. Any free protein binding sites on thedish are then covered by incubating with a non-specific protein likebovine serum albumin (BSA). Next, the monoclonal antibody is incubatedin the dish during which time the monoclonal antibodies attach to anyFhm proteins attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to Fhm.Unattached reporter antibody is then washed out. Peroxidase substratesare then added to the dish and the amount of color developed in a giventime period is a measurement of the amount of Fhm protein present in agiven volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to Fhmare attached to a solid support and labeled Fhm and a sample derivedfrom the host are passed over the solid support and the amount of labeldetected, for example, by liquid scintillation chromotagraphy, can becorrelated to a quantity of Fhm in the sample. In addition, a sandwichimmuno-assay as described above may also be carried out to quantify theamount of Fhm in a biological sample.

The sequences of the present invention are also valuable for chromosomeidentification and mapping. The sequence can be specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome wherein a gene can be localized. Few chromosomemarking reagents based on actual sequence data (repeat polymorphisms)are presently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the3′-untranslated region of the sequence is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified, fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map Fhm to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of genomic clones orclones from which the express sequence tag (EST) was derived, and thelonger the better. For example, 2,000 bp is good, 4,000 is better, andmore than 4,000 is probably not necessary to get good results areasonable percentage of the time. For a review of this technique seeVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The nucleic acid molecule(s) of the present invention are also useful asanti-sense inhibitors of Fhm expression. Such inhibition may be effectedby nucleic acid molecules which are complementary to and hybridize toexpression control sequences (triple helix formation) or to Fhm mRNA.Anti-sense probes may be designed by available techniques using thesequence of Fhm disclosed herein. Anti-sense inhibitors provideinformation relating to the decrease or absence of a Fhm polypeptide ina cell or organism.

The nucleic acid molecules of the invention may be used for genetherapy. Nucleic acid molecules which express Fhm in vivo provideinformation relating to the effects of the polypeptide in cells ororganisms. Fhm nucleic acid molecules, fragments, and/or derivativesthat do not themselves encode biologically active polypeptides may beuseful as hybridization probes in diagnostic assays to test, eitherqualitatively or quantitatively, for the presence of Fhm DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

Fhm polypeptide fragments, variants, and/or derivatives, whetherbiologically active or not, are useful for preparing antibodies thatbind to an Fhm polypeptide. The antibodies may be used for in vivo andin vitro diagnostic purposes, such as in labeled form to detect thepresence of Fhm polypeptide in a body fluid or cell sample. Theantibodies may bind to an Fhm polypeptide so as to diminish or block atleast one activity characteristic of an Fhm polypeptide, or may bind toa polypeptide to increase an activity.

Assaying for Modulators of Fhm Polypeptide Activity:

In some situations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity of Fhmpolypeptide. Natural or synthetic molecules that modulate Fhmpolypeptide can be identified using one or more of the screening assays,such as those described herein. Such molecules may be administeredeither in an ex vivo manner, or in an in vivo manner by local orintravenous (iv) injection, or by oral delivery, implantation device, orthe like.

“Test molecule(s)” refers to the molecule(s) that is/are underevaluation for the ability to modulate (i.e., increase or decrease) theactivity of an Fhm polypeptide. Most commonly, a test molecule willinteract directly with an Fhm polypeptide. However, it is alsocontemplated that a test molecule may also modulate Fhm polypeptideactivity indirectly, such as by affecting Fhm gene expression, or bybinding to an Fhm binding partner (e.g., receptor). In one embodiment, atest molecule will bind to an Fhm polypeptide with an affinity constantof at least about 10⁻⁶ M, preferably about 10⁻⁸ M, more preferably about10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

Methods for identifying compounds which interact with Fhm receptorpolypeptides are encompassed by the invention. In certain embodiments, aFhm receptor polypeptide is incubated with a test molecule underconditions which permit interaction of the test molecule to the receptorpolypeptide, in the presence or absence of bioactive Fhm, and the extentof the interaction can be measured. The test molecules can be screenedin a substantially purified form or in a crude mixture.

In certain embodiments, a Fhm polypeptide agonist or antagonist may be aprotein, peptide, carbohydrate, lipid, or small molecular weightmolecule which interacts with Fhm polypeptide to regulate its activity.Molecules which regulate Fhm polypeptide expression include nucleicacids which are complementary to nucleic acids encoding an Fhmpolypeptide, or are complementary to nucleic acids acid sequences whichdirect or control the expression of Fhm polypeptide, and which act asanti-sense regulators of expression.

The measurement of the interaction of test molecules with putative Fhmreceptor polypeptide(s) in the presence or absence of Fhm ligand may becarried out in several formats, including cell-based binding assays,membrane binding assays, solution-phase assays and immunoassays. Ingeneral, test molecules are incubated with a putative Fhm receptorpolypeptide for a specified period of time and Fhm polypeptide activityis determined by one or more assays measuring biological activity.

The interaction of test molecules with Fhm polypeptides may also beassayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of Fhm polypeptidescontaining epitope tags as described herein may be used in immunoassays.

Homogeneous assay technologies for radioactivity (SPA; Amersham) andtime resolved fluorescence (HTRF, Packard) can also be implemented.Binding can be detected by labeling with radioactive isotopes (¹²⁵I,³⁵S, ³H), fluorescent dyes (fluorescein), lanthanides such as Europeum(Eu³⁻) chelates or cryptates, orbipyridyl-ruthenium (Ru²⁻) complexes. Itis understood that the choice of a labeled probe will depend upon thedetection system used. Alternatively, Fhm or putative Fhm agonists orantagonists may be modified with an unlabeled epitope tag (e.g., biotin,peptides, His6, myc, Fc) and bound to proteins such as streptavidin,anti-peptide or anti-protein antibodies which have a detectable label asdescribed above.

Binding of test molecules to putative Fhm receptor polypeptides may alsobe assayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of putative Fhm-receptorpolypeptide(s) containing epitope tags as described above may be used insolution and immunoassays.

In one embodiment, modulators of the Fhm-ligand may be a protein,peptide, carbohydrate, lipid or small molecular weight molecule.Potential protein antagonists of Fhm include antibodies which bind toactive regions of the polypeptide and inhibit or eliminate binding ofFhm to its putative receptor. Molecules which regulate Fhm polypeptideexpression may include nucleic acids which are complementary to nucleicacids encoding a Fhm polypeptide, or are complementary to nucleic acidssequences which direct or control expression of polypeptide, and whichact as anti-sense regulators of expression.

In the event that Fhm polypeptides display biological activity throughan interaction with a binding partner (e.g., a receptor), a variety ofin vitro assays may be used to measure binding of Fhm polypeptide to acorresponding binding partner (such as a selective binding agent orlignad). These assays may be used to screen test molecules for theirability to increase or decrease the rate and/or the extent of binding ofa Fhm polypeptide to its binding partner. In one assay, Fhm polypeptideis immobilized in the wells of a microtiter plate. Radiolabeled Fhmbinding partner (for example, iodinated Fhm binding partner) and thetest molecule(s) can then be added either one at a time (in eitherorder) or simultaneously to the wells. After incubation, the wells canbe washed and counted (using a scintillation counter) for radioactivityto determine the extent of binding to which the binding partner bound toFhm polypeptide. Typically, the molecules will be tested over a range ofconcentrations, and a series of control wells lacking one or moreelements of the test assays can be used for accuracy in the evaluationof the results. An alternative to this method involves reversing the“positions” of the proteins, i.e. immobilizing Fhm binding partner tothe microtiter plate wells, incubating with the test molecule andradiolabeled Fhm and determining the extent of Fhm binding (see, forexample, Chapter 18 of Current Protocols in Molecular Biology, Ausubelet al., eds., John Wiley & Sons, New York, N.Y., 1995).

As an alternative to radiolabeling, a Fhm polypeptide or its bindingpartner may be conjugated to biotin and the presence of biotinylatedprotein can then be detected using streptavidin linked to an enzyme,such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), thatcan be detected colorometrically, or by fluorescent tagging ofstreptavidin. An antibody directed to an Fhm polypeptide or to an Fhmbinding partner and is conjugated to biotin may also be used and can bedetected after incubation with enzyme-linked streptavidin linked to APor HRP

A Fhm polypeptide and a Fhm binding partner can also be immobilized byattachment to agarose beads, acrylic beads or other types of such inertsolid phase substrates. The substrate-protein complex can be placed in asolution containing the complementary protein and the test compound.After incubation, the beads can be precipitated by centrifugation, andthe amount of binding between an Fhm polypeptide and its binding partnercan be assessed using the methods described above. Alternatively, thesubstrate-protein complex can be immobilized in a column and the testmolecule and complementary protein passed over the column. Formation ofa complex between an Fhm polypeptide and its binding partner can then beassessed using any of the techniques described herein, i.e.,radiolabeling, antibody binding, or the like.

Another in vitro assay that is useful for identifying a test moleculewhich increases or decreases the formation of a complex between a Fhmbinding protein and a Fhm binding partner is a surface plasmon resonancedetector system such as the BIAcore assay system (Pharmacia, Piscataway,N.J.). The BIAcore system may be carried out using the manufacturer'sprotocol. This assay essentially involves the covalent binding of eitherFhm or a Fhm binding partner to a dextran-coated sensor chip which islocated in a detector. The test compound and the other complementaryprotein can then be injected either simultaneously or sequentially intothe chamber containing the sensor chip. The amount of complementaryprotein binds can be assessed based on the change in molecular masswhich is physically associated with the dextran-coated side of thesensor chip; the change in molecular mass can be measured by thedetector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between a Fhm polypeptide and a Fhm bindingpartner complex. In these cases, the assays described herein can bereadily modified by adding such additional test compound(s) eithersimultaneous with, or subsequent to, the first test compound. Theremainder of the steps in the assay are as set forth herein.

In vitro assays such as those described herein may be usedadvantageously to screen rapidly large numbers of compounds for effectson complex formation by Fhm and Fhm binding partner. The assays may beautomated to screen compounds generated in phage display, syntheticpeptide and chemical synthesis libraries.

Compounds which increase or decrease the formation of a complex betweena Fhm polypeptide and a Fhm binding partner may also be screened in cellculture using cells and cell lines expressing either Fhm or Fhm bindingpartner. Cells and cell lines may be obtained from any mammal, butpreferably will be from human or other primate, canine, or rodentsources. The binding of an Fhm polypeptide to cells expressing Fhmbinding partner at the surface is evaluated in the presence or absenceof test molecules and the extent of binding may be determined by, forexample, flow cytometry using a biotinylated antibody to an Fhm bindingpartner. Cell culture assays may be used advantageously to furtherevaluate compounds that score positive in protein binding assaysdescribed herein.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase the expression ofthe Fhm gene. In certain embodiments, the amount of Fhm polypeptide thatis produced may be measured after exposure of the cell culture to thedrug candidate. In certain embodiments, one may detect the actual impactof the drug candidate on the cell culture. For example, theoverexpression of a particular gene may have a particular impact on thecell culture. In such cases, one may test a drug candidate's ability toincrease or decrease the expression of the gene or its ability toprevent or inhibit a particular impact on the cell culture. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct in a cell culture.

P38 Inhibitors

A new approach to intervention between the extracellular stimulus andthe secretion of IL-1 and TNFα from the cell involves blocking signaltransduction through inhibition of a kinase which lies on the signalpathway. One example is through inhibition of P-38 (also called “RK” or“SAPK-2”, Lee et al., Nature, 372:739, 1994), a known ser/thr kinase(clone reported in Han et al., Biochimica Biophysica Acta, 1265:224-227,1995). A linear relationship has been shown for effectiveness in acompetitive binding assay to P-38, and the same inhibitor diminishingthe levels of IL-1 secretion from monocytes following LPS stimulation.Following LPS stimulation of monocytes, the levels of messenger RNA forTNF-α have been shown to increase 100 fold, but the protein levels ofTNF-α are increased 10,000 fold. Thus, a considerable amplification ofthe TNF signaling occurs at the translational level. Following LPSstimulation of monocytes in the presence of a P-38 inhibitor, the levelsof mRNA are not affected, but the levels of final TNF protein aredramatically reduced (up to 80-90% depending on the effectiveness of theP-38 inhibitor). Thus, the above experiments lend strong support to theconclusion that inhibition of P-38 leads to diminished translationalefficiency. Further evidence that TNFa is under translational control isfound in the deletion experiments of Beutler et al. and Lee, whereinsegments of 3′ untranslated mRNA (3′ UTR) are removed resulting in hightranslational efficiency for TNFα. More importantly, the P-38 inhibitorsdid not have an effect on the level of TNFα (i.e., translationalefficiency) when the appropriate segments of TNFα mRNA are deleted.Thus, the correlative data between the level of binding of inhibitors toP-38 and the diminished IL-1 and TNFα levels following LPS stimulationwith the same inhibitors, plus the above biochemical evidence regardingthe effect of P-38 inhibitors on translational efficiency of both TNFαand IL-1 make a strong cause and effect relationship. The role of P-38in the cell is still being delineated; so therefore, other beneficialeffects regarding inflammatory diseases or other disease states obtainedfrom its inhibition maybe forthcoming.

Elevated levels of TNFα and/or IL-1 may contribute to the onset,etiology, or exacerbate a number of disease states, including, but notlimited to: rheumatoid arthritis; osteoarthritis; rheumatoidspondylitis; gouty arthritis; inflammatory bowel disease; adultrespiratory distress syndrome (ARDS); psoriasis; Crohn's disease;allergic rhinitis; ulcerative colitis; anaphylaxis; contact dermatitis;asthma; antiviral therapy including those viruses sensitive to TNFαinhibition—HIV-1, HIV-2, HIV-3, cytomegalovirus (CMV), influenza,adenovirus, and the herpes viruses including HSV-1, HSV-2, and herpeszoster; muscle degeneration; cachexia; Reiter's syndrome; type IIdiabetes; bone resorption diseases; graft vs. host reaction; ischemiareperfusion injury; atherosclerosis; brain trauma; Alzheimer's disease;multiple sclerosis; cerebral malaria; sepsis; septic shock; toxic shocksyndrome; fever and mylagias due to infection.

Substituted imidazole, pyrrole, pyridine, pyrimidine and the likecompounds have been described for use in the treatment of cytokinemediated diseases by inhibition of proinflammatory cytokines, such asIL-1, IL-6, IL-8 and TNF. Substituted imidazoles for use in thetreatment of cytokine mediated diseases have been described in U.S. Pat.No. 5,593,992; WO 93/14081; WO 97/18626; WO 96/21452; WO 96/21654; WO96/40143; WO 97/05878; WO 97/05878; (each of which is incorporatedherein by reference in its entirety). Substituted imidazoles for use inthe treatment of inflammation has been described in U.S. Pat. No.3,929,807 (which is incorporated herein by reference in its entirety).Substituted pyrrole compounds for use in the treatment of cytokinemediated diseases have been described in WO 97/05877; WO 97/05878; WO97/16426; WO 97/16441; and WO 97/16442 (each of which is incorporatedherein by reference in its entirety). Substituted aryl and heteroarylfused pyrrole compounds for use in the treatment of cytokine mediateddiseases have been described in WO 98/22457 (which is incorporatedherein by reference in its entirety). Substituted pyridine, pyrimidine,pyrimidinone and pyridazine compounds for use in the treatment ofcytokine mediated diseases have been described in WO 98/24780; WO98/24782; WO 99/24404; and WO 99/32448 (each of which is incorporatedherein by reference in its entirety).

Internalizing Proteins

The TAT protein sequence (from HIV) can be used to internalize proteinsinto a cell by targeting the lipid bi-layer component of the cellmembrane. See e.g., Falwell et al., Proc. Natl. Acad. Sci., 91: 664-668,1994. For example, an 11 amino acid sequence (YGRKKRRQRRR; SEQ ID NO:22) of the HIV TAT protein (termed the “protein transduction domain”, orTAT PDT) has been shown to mediate delivery of large bioactive proteinssuch as β-galactosidase and p27Kip across the cytoplasmic membrane andthe nuclear membrane of a cell. See Schwarze et al., Science, 285:1569-1572, 1999; and Nagahara et al., Nature Medicine, 4: 1449-1452,1998. Schwartze et al. (Science, 285: 1569-72, 1999) demonstrated thatcultured cells acquired β-gal activity when exposed to a fusion of theTAT PDT and β-galactosidase. Injection of mice with the TAT-β-gal fusionproteins resulted in β-gal expression in a number of tissues, includingliver, kidney, lung, heart, and brain tissue.

It will thus be appreciated that the TAT protein sequence may be used tointernalize a desired protein or polypeptide into a cell. In the contextof the present invention, the TAT protein sequence can be fused toanother molecule such as a Fhm antagonist (i.e.: anti-Fhm selectivebinding agent or small molecule) and administered intracellularly toinhibit the activity of the Fhm molecule. Where desired, the Fhm proteinitself, or a peptide fragment or modified form of Fhm, may be fused tosuch a protein transducer for administrating to cells using theprocedures, described above.

Therapeutic Uses

Members of the TNF ligand family have been implicated in mediation of anumber of diseases. The pleiotropic nature of TNF and related ligandfamily members prevents generalization about whether a particularpolypeptide is beneficial or injurious. It is clear that in someinstances, the local effects of TNF and other members of the TNF-ligandfamily of cytokines improve host defense mechanisms by mobilizingsubstrate, increasing immune cell function, stimulating inflammation,and in killing cancer cells. However, in other cases the toxicity of TNFand related cytokines may cause disease by mediating shock, tissueinjury, or catabolic injury. There are many diseases wherein injury thatis mediated by members of the TNF ligand family may be treated orameliorated by the administration of soluble forms of members of theTNF-receptor gene family or TNF-like ligand molecules. These diseasesinclude acquired-immunodeficiency syndrome (AIDS), anemia, autoimmunediseases, cachexia, cancer, cerebral malaria, diabetes mellitus,disseminated intravascular coagulopathy, erythryoid sick syndrome,hemorrhagic shock, hepatitis, insulin resistance, leprosy, leukemia,lymphoma, meningitis, multiple sclerosis, myocardial ischaemia, obesity,rejection of transplanted organs, rheumatoid arthritis, septic shocksyndrome, stroke, adult respiratory distress syndrome (ARDS),tuberculosis, and a number of viral diseases.

Fhm Compositions and Administration

Pharmaceutical compositions of Fhm polypeptides are within the scope ofthe present invention for prophylactic and therapeutic treatment ofhumans and animals for indications resulting from abnormal expression ofFhm or where it is determined that administration of Fhm polypeptidewill result in the amelioration or cure of the indications. Suchcompositions may comprise a therapeutically effective amount of a Fhmpolypeptide and/or its binding partner, or therapeutically activefragment(s), variant(s), or derivative(s) thereof in admixture with apharmaceutically acceptable additives and/or carriers. Suitableformulation materials or pharmaceutically acceptable agents include, butare not limited to, antioxidants, preservatives, colors, flavoring, anddiluting agents, emulsifying agents, suspending agents, solvents,fillers, bulking agents, buffers, delivery vehicles, diluents,excipients, and/or pharmaceutical adjuvants. Typically, a therapeuticcompound containing Fhm polypeptide(s) will be administered in the formof a composition comprising purified polypeptide, fragment(s),variant(s), or derivative(s) in conjunction with one or morephysiologically acceptable carriers, excipients, or diluents. Forexample, a suitable vehicle may be water for injection, physiologicalsolution, or artificial cerebrospinal fluid possibly supplemented withother materials common in compositions for parenteral delivery.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. Preferably, the product is formulated as alyophilizate using appropriate excipients (e.g., sucrose). Otherstandard carriers, diluents, and excipients may be included as desired.Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5,or acetate buffer of about pH 4.0-5.5, which may further includesorbitol or a suitable substitute therefor. The pH of the solutionshould also be selected based on the relative solubility of Fhm atvarious pHs.

The primary solvent in a composition may be either aqueous ornon-aqueous in nature. In addition, the vehicle may contain otherformulation materials for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, isotonicity, rate ofdissolution, or odor of the formulation. Similarly, the composition maycontain additional formulation materials for modifying or maintainingthe rate of release of Fhm protein, or for promoting the absorption orpenetration of Fhm protein.

Compositions comprising the Fhm polypeptide compositions can beadministered parentally. Alternatively, the compositions may beadministered intravenously or subcutaneously. When systemicallyadministered, the therapeutic compositions for use in this invention maybe in the form of a pyrogen-free, parentally acceptable aqueoussolution. The preparation of such pharmaceutically acceptable proteinsolutions, with due regard to pH, isotonicity, stability and the like,is within the skill of the art.

Therapeutic formulations of Fhm polypeptide compositions useful forpracticing the present invention may be prepared for storage by mixingthe selected composition having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company, 1990) in the form of a lyophilized cake or anaqueous solution.

Acceptable carriers, excipients or stabilizers are nontoxic torecipients and are preferably inert at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, or otherorganic acids; antioxidants such as ascorbic acid; low molecular weightpolypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, pluronics orpolyethylene glycol (PEG).

An effective amount of the Fhm polypeptide(s) composition to be employedtherapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which the composition is beingused, the route of administration (e.g., whether it is administeredlocally or systemically), and the condition of the patient (e.g.,patient's general health, anaureuesis, age, weight, sex). It isessential, when determining the therapeutically effective dose, to takeinto account the quantity of Fhm or other members of the TNF family thatare responsible for the disease. Basically, it can be assumed that foreffective treatment of a disease triggered by the over expression ofcytokine(s) such as Fhm, at least the same molar amount of the Fhmpolypeptide(s) is required, and possibly a multiple excess might beneeded, although less may be needed depending on the nature of thereceptor and the nature of its interaction with Fhm. Accordingly, itwill be necessary for the therapist to titer the dosage and/or in vivomodify the route of administration as required to obtain the optimaltherapeutic effect. A typical daily dosage may range from about 0.1mg/kg to up to 100 mg/kg or more, depending on the factors mentionedabove. Typically, a clinician will administer the composition until adosage is reached that achieves the desired effect. The composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of Fhm polypeptide) overtime, or as a continuous infusion via implantation device or catheter.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, the type of disorder under treatment, the age and generalhealth of the recipient, will be able to ascertain proper dosing.

The Fhm polypeptide composition to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes, Where the composition is lyophilized,sterilization using thes method may be conducted either prior to orfollowing lyophilization and reconstitution. The composition forparenteral administration ordinarily will be stored in lyophilized formor in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle. Once thepharmaceutical composition has been formulated, it may be stored insterile vials as a solution, suspension, gel, emulsion, solid, or as adehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

Effective administration forms, such as (1) slow-release formulations,(2) inhalant mists, or (3) orally active formulations are alsoenvisioned. Pharmaceutical compositions comprising thereapeuticallyeffective dose of the Fhm polypeptide also may be formulated forparenteral administration. Such parenterally administered therapeuticcompositions are typically in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising Fhm in a pharmaceuticallyacceptable vehicle. The Fhm pharmaceutical compositions also may includeparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc. or the introduction of Fhm into liposomes.Hyaluronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation.

A particularly suitable vehicle for parenteral injection is steriledistilled water in which Fhm is formulated as a sterile, isotonicsolution, properly preserved. Yet another preparation may involve theformulation of Fhm with an agent, such as injectable microspheres,bio-erodible particles or beads, or liposomes, that provides for thecontrolled or sustained release of the protein product which may then bedelivered as a depot injection. Other suitable means for theintroduction of Fhm include implantable drug delivery devices whichcontain the Fhm and/or its binding partner.

The preparations of the present invention may include other components,for example parenterally acceptable preservatives, tonicity agents,cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, antioxidants and surfactants, as are well known in theart. For example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol and the like. Suitable preservatives include, but are notlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsare for example glycerin, propylene glycol and polyethylene glycol.Suitable complexing agents are for example caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal and the like. The bufferscan be conventional buffers such as borate, citrate, phosphate,bicarbonate, or Tris-HCl.

The formulation components are present in concentration that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired Fhm molecule in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which an Fhm molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), or beads or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered via a depot injection. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

A pharmaceutical composition may be formulated for inhalation. Forexample, Fhm may be formulated as a dry powder for inhalation. Fhmpolypeptides or Fhm nucleic acid molecule inhalation solutions may alsobe formulated with a d propellant for aerosol delivery. In yet anotherembodiment, solutions may be nebulized. Pulmonary administration isfurther described in PCT Application No. PCT/US94/01875, which describespulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations containing Fhmpolypeptide(s) may be administered orally. In one embodiment, the Fhmligand which is administered in this fashion may be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the Fhm polypeptide. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

Another pharmaceutical composition may involve an effective quantity ofFhm polypeptide in a mixture with non-toxic excipients which aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or other appropriate vehicle, solutions can be preparedin unit dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional Fhm-polypeptide pharmaceutical compositions will be evidentto those skilled in the art, including formulations involvingFhm-polypeptide in sustained or controlled delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, PCT Application No.PCT/US93/00829 which describes the controlled release porous polymericmicroparticles for the delivery of pharmaceutical compositions.Additional examples include semipermeable polymer matrices in the formof shaped articles e.g., films or microspheres.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

The effective amount of an Fhm pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the Fhmmolecule is being used, the route of administration, and the size (bodyweight, body surface or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 mg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 mg/kg up to about 100 mg/kg;or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the Fhm molecule in the formulation used. Typically, a clinician willadminister the composition until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as two or more doses (which may or may not contain thesame amount of the desired molecule) over time, or as a continuousinfusion via an implantation device or catheter. Further refinement ofthe appropriate dosage is routinely made by those of ordinary skill inthe art and is within the ambit of tasks routinely performed by them.Appropriate dosages may be ascertained through use of appropriatedose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes, or routes; by sustained releasesystems or by implantation devices. Where desired, the compositions maybe administered by bolus injection or continuously by infusion, or byimplantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or another appropriatematerial on to which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

One may further administer the present pharmaceutical compositions bypulmonary administration, see, e.g., International Publication No: WO94/20069, which discloses pulmonary delivery of chemically modifiedproteins, herein incorporated by reference. For pulmonary delivery, theparticle size should be suitable for delivery to the distal lung. Forexample, the particle size may be from 1 mm to 5 mm, however, largerparticles may be used, for example, if each particle is fairly porous.Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which receptor polypeptide has beenabsorbed or encapsulated. Where an implantation device is used, thedevice may be implanted into any suitable tissue or organ, and deliverymay be directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

Fhm-ligand polypeptide(s) and/or its binding partner may also beadministered in a sustained release formulation or preparation. Suitablepolymer compositions preferably have intrinsic and controllablebiodegradability so that they persist for about a week to about sixmonths; are non-toxic containing no significant toxic monomers anddegrading into non-toxic components; are biocompatible, are chemicallycompatible with substances to be delivered, and tend not to denature theactive substance; are sufficiently porous to allow the incorporation ofbiologically active molecules and their subsequent liberation from thepolymer by diffusion, erosion or a combination thereof; are able toremain at the site of the application by adherence or by geometricfactions, such as being formed in place or softened and subsequentlymolded or formed into microparticles which are trapped at a desiredlocation; are capable of being delivered by techniques of minimuminvasivity such as by catheter, laparoscope or endoscope. Sustainedrelease matrices include polyesters, hydrogels, polylactides (U.S. Pat.No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277, 1981 and Langer, Chem. Tech., 12: 98-105, 1982), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). Sustained-release compositions also may include liposomes,which can be prepared by any of several methods known in the art (e.g.,Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692. 1985; EP36,676; EP 88,046; EP 143,949, incorporated herein by reference).

The Fhm polypeptides, variants, derivatives or fragments thereof, may beemployed alone, together, or in combination with other pharmaceuticalcompositions. The Fhm polypeptides, fragments, variants, and derivativesmay be used in combination with cytokines, cytokine inhibitors, growthfactors, antibiotics, anti-inflammatories, and/or chemotherapeuticagents as is appropriate for the indication being treated

In some cases, it may be desirable to use Fhm polypeptide pharmaceuticalcompositions in an ex vivo manner. In such instances, cells, tissues, ororgans that have been removed from the patient are exposed to Fhmpolypeptide pharmaceutical compositions after which the cells, tissuesand/or organs are subsequently implanted back into the patient.

In other cases, a Fhm polypeptide can be delivered by implanting intopatients certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides, fragments, variants, or derivatives. Such cells may beanimal or human cells, and may autologous, heterologous or xenogeneic.Optionally, the cells may be immortalized. In order to decrease thechance of an immunological response, it is preferred that the cells maybe encapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

Methods used for membrane encapsulation of cells are familiar to theskilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627, incorporated herein by reference. A system for encapsulatingliving cells is described in International Publication No: WO 91/10425.Techniques for formulating a variety of other sustained or controlleddelivery means, such as liposome carriers, bio-erodible particles orbeads, are also known to those in the art, and are described, forexample, in U.S. Pat. No. 5,653,975, incorporated herein by reference.The cells, with or without encapsulation, may be implanted into suitablebody tissues or organs of the patient.

As discussed herein, it may be desirable to treat isolated cellpopulations such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like, add as appropriate with one or moreFhm polypeptides, variants, derivatives and/or fragments. This can beaccomplished by exposing the isolated cells to the polypeptide, variant,derivative, or fragment directly; where it is in a form that ispermeable to the cell membrane.

The present invention relates to improved methods for both the in vitroproduction of therapeutic proteins and for the production and deliveryof therapeutic proteins by gene therapy.

Homologous Recombination

It is further envisioned that Fhm protein may be produced by homologousrecombination, or with recombinant production methods utilizing controlelements introduced into cells already containing DNA encoding Fhm. Forexample, homologous recombination methods may be used to modify a cellthat contains a normally transcriptionally silent Fhm gene, or underexpressed gene, and thereby produce a cell which expressestherapeutically efficacious amounts of Fhm. Homologous recombination isa technique originally developed for targeting genes to induce orcorrect mutations in transcriptionally active genes (Kucherlapati, Prog.in Nucl. Acid Res. and Mol. Biol., 36:301, 1989). The basic techniquewas developed as a method for introducing specific mutations intospecific regions of the mammalian genome (Thomas et al., Cell,44:419-428, 1986; Thomas and Capecchi, Cell, 51:503-512, 1987;Doetschman et al., Proc. Natl. Acad. Sci., 85:8583-8587, 1988) or tocorrect specific mutations within defective genes (Doetschman et al.,Nature, 330:576-578, 1987). Exemplary homologous recombinationtechniques are described in U.S. Pat. No. 5,272,071, EP Publication No:91 90 3051, EP Publication No. 505 500; PCT/US90/07642, InternationalPublication No: WO 91/09955, incorporated herein by reference.

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA, into which insertion of the sequence is sought. Small pieces oftargeting DNA that are complementary to a specific region of the genomeare put in contact with the parental strand during the DNA replicationprocess. It is a general property of DNA that has been inserted into acell to hybridize, and therefore, recombine with other pieces ofendogenous DNA through shared homologous regions. If this complementarystrand is attached to an oligonucleotide that contains a mutation or adifferent sequence or an additional nucleotide, it too is incorporatedinto the newly synthesized strand as a result of the recombination. As aresult of the proofreading function, it is possible for the new sequenceof DNA to serve as the template. Thus, the transferred DNA isincorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA which mayinteract with or control the expression of a Fhm polypeptide, e.g.flanking sequences. For example, a promoter/enhancer element, asuppresser, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredFhm polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of Fhm protein maybe achieved not by transfection of DNA that encodes the Fhm gene itself,but rather by the use of targeting DNA (containing regions of homologywith the endogenous gene of interest) coupled with DNA regulatorysegments that provide the endogenous gene sequence with recognizablesignals for transcription of a Fhm protein.

In an exemplary method, expression of a desired targeted gene in a cell(i.e., a desired endogenous cellular gene) is altered by theintroduction, by homologous recombination into the cellular genome at apreselected site, by the introduction of DNA which includes at least aregulatory sequence, an exon and a splice donor site. These componentsare introduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

Altered gene expression, as described herein, encompasses activating (orcausing to be expressed) a gene which is normally silent (unexpressed)in the cell as obtained, as well as increasing the expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained. The embodiment s further encompass changing the pattern ofregulation or induction such that it is different from the pattern ofregualtion or induction that occurs in the cell as obtained, andreducing (including eliminating) expression of a gene which is expressedin the cell as obtained.

One method by which homologous recombination can be used to increase, orcause, Fhm polypeptide production from a cell's endogenous Fhm geneinvolves first using homologous recombination to place a recombinationsequence from a site-specific recombination system (e.g., Cre/loxP,FLP/FRT) (see, Sauer, Current Opinion In Biotechnology, 5:521-527, 1994;and Sauer, Methods In Enzymology, 225:890-900, 1993) upstream (that is,5′ to) of the cell's endogenous genomic Fhm polypeptide coding region. Aplasmid containing a recombination site homologous to the site that wasplaced just upstream of the genomic Fhm polypeptide coding region isintroduced into the modified cell line along with the appropriaterecombinase enzyme. This recombinase enzyme causes the plasmid tointegrate, via the plasmid's recombination site, into the recombinationsite located just upstream of the genomic Fhm polypeptide coding regionin the cell line (Baubonis and Sauer, Nucleic Acids Res., 21:2025-2029,1993; and O'Gorman et al., Science, 251:1351-1355, 1991). Any flankingsequences known to increase transcription (e.g., enhancer/promoter,intron, or translational enhancer), if properly positioned in thisplasmid, would integrate in such a manner as to create a new or modifiedtranscriptional unit resulting in de novo or increased Fhm polypeptideproduction from the cell's endogenous Fhm gene.

A further method to use the cell line in which the site-specificrecombination sequence has been placed just upstream of the cell'sendogenous genomic Fhm polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, or translocation) (Sauer,Current Opinion In Biotechnology, supra, 1994; and Sauer, Methods InEnzymology, supra, 1993) that would create a new or modifiedtranscriptional unit resulting in de novo or increased Fhm polypeptideproduction from the cell's endogenous Fhm gene.

An additional approach for increasing, or causing, the expression of Fhmpolypeptide from a cell's endogenous Fhm gene involves increasing, orcausing, the expression of a gene or genes (e.g., transcription factors)and/or decreasing the expression of a gene or genes (e.g.,transcriptional repressors) in a manner which results in de novo orincreased Fhm polypeptide production from the cell's endogenous Fhmgene. This method includes the introduction of a non-naturally occurringpolypeptide (e.g., a polypeptide comprising a site specificsite-specific DNA binding domain fused to a transcriptional factordomain) into the cell such that de novo or increased Fhm polypeptideproduction from the cell's endogenous Fhm gene results.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) on or more targetingsequence; (b) a regulatory sequence; (c) an exon; and (d) an unpairedsplice-donor site. The targeting sequence in the DNA construct directsthe integration of elements (a)-(d) into a target gene in a cell suchthat the elements (b)-(d) are operatively linked to sequences of theendogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequence, (b) a regulatory sequence,(c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

If the sequence of a particular gene is known, such as the nucleic acidsequence of Fhm presented herein, a piece of DNA that is complementaryto a selected region of the gene can be synthesized or otherwiseobtained, such as by appropriate restriction of the native DNA atspecific recognition sites bounding the region of interest. This pieceserves as a targeting sequence(s) upon insertion into the cell and willhybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be incorporated into the newly synthesized daughter strand ofDNA. The present invention, therefore, includes nucleotides encodingFhm-polypeptide(s), which nucleotides may be used as targetingsequences.

Alternatively, gene therapy can be employed as described below.

Fhm Cell Therapy and Gene Therapy

Fhm cell therapy, e.g., the implantation of cells producing Fhm, is alsoencompassed by the present invention. This embodiment involvesimplanting cells capable of synthesizing and secreting a biologicallyactive form of the soluble Fhm. Such soluble Fhm polypeptide producingcells may be cells that are natural producers of Fhm or may berecombinant cells whose ability to produce Fhm has been augmented bytransformation with a gene encoding the desired Fhm molecule or with agene augmenting the expression of Fhm, polypeptide. Such a modificationmay be accomplished by means of a vector suitable for delivering thegene as well as promoting its expression and secretion. In order tominimize a potential immunological reaction in patients beingadministered a Fhm polypeptide as may occur with the adminstration of apolypeptide of a foreign species, it is preferred that the natural cellsproducing Fhm be of human origin and produce human Fhm. Likewise, it ispreferred that the recombinant cells producing Fhm be transformed withan expression vector containing a gene encoding a human Fhm polypeptide.

Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow release of Fhm polypeptide but that prevent thedestruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce Fhm polypeptides ex vivo,may be implanted directly into the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be accomplished without undue experimentation. For example,Baetge et al. (WO 95105452 and PCT/US94/09299) describe membranecapsules containing genetically engineered cells for the effectivedelivery of biologically active molecules. The capsules arebiocompatible and are easily retrievable. The capsules are biocompatibleand are easily retrievable. The capsules encapsulate cells transfectedwith recombinant DNA molecules comprising DNA sequences coding forbiologically active molecules operatively linked to promoters that arenot subject to down-regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538, 5,011,472, and 5,106,627, incorporated hereinby reference. A system for encapsulating living cells is described inInternational Application WO 91/10425 of Aebischer et al., InternationalApplication No. WO 91/10470 of Aebischer et al.; Winn et al., Exper.Neurol., 113:322-329, 1991, Aebischer et al. Exper. Neurol., 111:269-275, 1991; and Tresco et al., ASAIO, 38:17-23, 1992, incorporatedherein by reference.

In vivo and in vitro gene therapy delivery of Fhm is also encompassed bythe present invention. In vivo gene therapy may be accomplished byintroducing the gene encoding Fhm into cells via local injection of apolynucleotide molecule or other appropriate delivery vectors. (Hefti,J. Neurobiology,. 25:1418-1435, 1994). For example, a polynucleotidemolecule encoding Fhm may be contained in an adeno-associated virusvector for delivery to the targeted cells (See for e.g., InternationalPublication No. WO 95/34670; International Application No.PCT/US95/07178). The recombinant adeno-associated virus (AAV) genometypically contains AAV inverted terminal repeats flanking a DNA sequenceencoding Fhm operably linked to functional promoter and polyadenylationsequences.

Alternative viral vectors include, but are not limited to, retrovirus,adenovirus, herpes simplex virus and papilloma virus vectors. U.S. Pat.No. 5,672,344 (issued Sep. 30, 1997, Kelley et al., University ofMichigan) describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 (issued Mar. 21, 1995, Anderson et al., Department of Healthand human Services) provides examples of a process for providing apatient with a therapeutic protein by the delivery of human cells whichhave been treated in vitro to insert a DNA segment encoding atherapeutic protein. Additional methods and materials for the practiceof gene therapy techniques are described in U.S. Pat. No. 5,631,236(issued May 20, 1997, Woo et al., Baylor College of Medicine) involvingadenoviral vectors; U.S. Pat. No. 5,672,510 (issued Sep. 30, 1997,Eglitis et al., Genetic Therapy, Inc.) involving retroviral vectors; andU.S. Pat. No. 5,635,399 (issued Jun. 3, 1997, Kriegler et al., ChironCorporation) involving retroviral vectors expressing cytokines.

Nonviral delivery methods include liposome-mediated transfer, naked DNAdelivery (direct injection), receptor-mediated transfer (ligand-DNAcomplex), electroporation, calcium phosphate precipitation andmicroparticle bombardment (e.g., gene gun). Gene therapy materials andmethods may also include inducible promoters, tissue-specificenhancer-promoters, DNA sequences designed for site-specificintegration, DNA sequences capable of providing a selective advantageover the parent cell, labels to identify transformed cells, negativeselection systems and expression control systems (safety measures),cell-specific binding agents (for cell targeting), cell-specificinternalization factors, transcription factors to enhance expression bya vector as well as methods of vector manufacture. Such additionalmethods and materials for the practice of gene therapy techniques aredescribed in U.S. Pat. No. 4,970,154 (issued Nov. 13, 1990, D. C. Chang,Baylor College of Medicine) electroporation techniques; InternationalApplication No. WO 9640958 (published 961219, Smith et al. BaylorCollege of Medicine) nuclear ligands; U.S. Pat. No. 5,679,559 (issuedOct. 21, 1997, Kim et al., University of Utah Research Foundation)concerning a lipoprotein-containing system for gene delivery; U.S. Pat.No. 5,676,954 (issued Oct. 14, 1997, K. L. Brigham, VanderbiltUniversity involving liposome carriers; U.S. Pat. No. 5,593,875 (issuedJan. 14, 1997. Wurm et al., Genentech, Inc.) concerning methods forcalcium phosphate transfection; and U.S. Pat. No. 4,945,050 (issued Jul.31, 1990, Sanford et al., Cornell Research Foundation) whereinbiologically active particles are propelled at cells at a speed wherebythe particles penetrate the surface of the cells and become incorporatedinto the interior of the cells. Expression control techniques includechemical induced regulation (e.g., International Application Nos. WO9641865 and WO 9731899), the use of a progesterone antagonist in amodified steroid hormone receptor system (e.g., U.S. Pat. No.5,364,791), ecdysone control systems (e.g., International ApplicationNo. WO 9637609), and positive tetracycline-controllable transactivators(e.g., U.S. Pat. Nos. 5,589,362; 5,650,298; and 5,654,168).

It is also contemplated that Fhm gene therapy or cell therapy canfurther include the delivery of a second protein. For example, the hostcell may be modified to express and release soluble forms of both Fhmand TNF-α, or Fhm and IL-1. Alternatively, the Fhm and TNF-α, or Fhm andIL-1, may be expressed in and released from separate cells. Such cellsmay be separately introduced into the patient or the cells may becontained in a single implantable device, such as the encapsulatingmembrane described above.

One manner in which gene therapy can be applied is to use the Fhm gene(either genomic DNA, cDNA, and/or synthetic DNA encoding a Fhmpolypeptide, or a fragment, variant, or derivative thereof) which may beoperably linked to a constitutive or inducible promoter to form a “genetherapy DNA construct”. The promoter may be homologous or heterologousto the endogenous Fhm gene, provided that it is active in the cell ortissue type into which the construct will be inserted. Other componentsof the gene therapy DNA construct may optionally include, as required,DNA molecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (for example, for cell targeting) cell-specificinternalization factors, and transcription factors to enhance expressionby a vector as well as factors to enable vector manufacture.

A gene therapy DNA construct can then be introduced into the patient'scells (either ex vivo or in vivo) using viral or non-viral vectors. Onemeans for introducing the gene therapy DNA construct. Certain vectors,such as retroviral vectors, will deliver the DNA construct to thechromosomal DNA of the cells, and the gene can integrate into thechromosomal DNA. Other vectors will function as episomes, and the genetherapy DNA construct will remain in the cytoplasm. The use of genetherapy vectors is described, for example, in U.S. Pat. Nos. 5,672,344;5,399,346; 5,631,236; and 5,635,399, incorporated herein by reference.

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the Fhm gene in the target cell. Such elementsare turned on in response to an appropriate effector. In this way, atherapeutic polypeptide can be expressed when desired. One conventionalcontrol means involves the use of small molecule dimerizers or rapalogs(as described in WO 9641865 (PCT/US96/099486); WO 9731898(PCT/US97/03137) and WO9731899 (PCT/US95/03157)WO 9731899(PCT/US95/03157)) used to dimerize chimeric proteins which contain asmall molecule-binding domain and a domain capable of initiatingbiological process, such as a DNA-binding protein or a transcriptionalactivation protein. The dimerization of the proteins can be used toinitiate transcription of the transgene.

An alternative regulation technology uses a method of storing proteinsexpressed from the gene of interest inside the cell as an aggregate orcluster. The gene of interest is expressed as a fusion protein thatincludes a conditional aggregation domain which results in the retentionof the aggregated protein in the endoplasmic reticulum. The storedproteins are stable and inactive inside the cell. The proteins can bereleased, however, by administering a drug (e.g., small molecule ligand)that removes the conditional aggregation domain and thereby specificallybreaks apart the aggregates or clusters so that the proteins may besecreted from the cell. See, Science 287:816-817, and 826-830 (2000).

Other suitable control means or gene switches include, but are notlimited to, the following systems. Mifepristone (RU486) is used as aprogesterone antagonist. The binding of a modified progesterone receptorligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors which thenpass into the nucleus to bind DNA. The ligand binding ligand-bindingdomain is modified to eliminate the ability of the receptor to bind tothe natural ligand. The modified steroid hormone receptor system isfurther described in U.S. Pat. No. 5,364,791; WO9640911, and WO9710337,WO 9640911 and WO 9710337.

Yet another control system uses ecdysone (a fruit fly steroid hormone)which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain/DNA-bindingdomain/ligand-binding domain to initiate transcription. The ecdysonesystem is further described in U.S. Pat. No. 5,514,578; WO9738117;WO9637609; WO 9738117; WO 9637609 and WO9303162.

Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758; 5,650,298 and 5,654,168.

Additional expression control systems and nucleic acid constructs aredescribed in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

In vivo gene therapy may be accomplished by introducing the geneencoding an Fhm polypeptide into cells via local injection of an Fhmnucleic acid molecule or by other appropriate viral or non-non-viraldelivery vectors. (Hefti, Neurobiology, 25:1418-1435, 1994). Forexample, a nucleic acid molecule encoding an Fhm polypeptide maybecontained in an adeno-associated virus (AAV) vector for delivery to thetargeted cells (e.g., Johnson, International Publication No. WO95/34670;and International Application No. PCT/US95/07178). The recombinant AAVgenome typically contains AAV inverted terminal repeats flanking a DNAsequence encoding an Fhm polypeptide operably linked to functionalpromoter and polyadenylation sequences.

Alternative suitable viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631,236 involving adenoviralvectors; U.S. Pat. No. 5,672,510 involving retroviral vectors; and U.S.Pat. No. 5,635,399 involving retroviral vectors expressing cytokines.

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include the useof inducible promoters, tissue-specific enhancer-promoters, DNAsequences designed for site-specific integration, DNA sequences capableof providing a selective advantage over the parent cell, labels toidentify transformed cells, negative selection systems and expressioncontrol systems (safety measures), cell-specific binding agents (forcell targeting), cell-specific internalization factors, andtranscription factors to enhance expression by a vector as well asmethods of vector manufacture. Such additional methods and materials forthe practice of gene therapy techniques are described in U.S. Pat. No.4,970,154 involving electroporation techniques; WO96/40958 involvingnuclear ligands; U.S. Pat. No. 5,679,559 describing alipoprotein-containing system for gene delivery; U.S. Pat. No. 5,676,954involving liposome carriers; U.S. Pat. No. 5,593,875 concerning methodsfor calcium phosphate transfection; and U.S. Pat. No. 4,945,050 whereinbiologically active particles are propelled at cells at a speed wherebythe particles penetrate the surface of the cells and become incorporatedinto the interior of the cells.

A means to increase endogenous Fhm polypeptide expression in a cell viagene therapy is to insert one or more enhancer elements into the Fhmpolypeptide promoter, where the enhancer element(s) can serve toincrease transcriptional activity of the Fhm polypeptides gene. Theenhancer element(s) used will be selected based on the tissue in whichone desires to activate the gene(s); enhancer elements known to conferpromoter activation in that tissue will be selected. For example, if aFhm gene encoding a Fhm polypeptide is to be “turned on” in T-cells, thelck promoter enhancer element may be used. Here, the functional portionof the transcriptional element to be added may be inserted into afragment of DNA containing the Fhm polypeptide promoter (and optionally,inserted into a vector, 5′ and/or 3′ flanking sequence(s), etc.) usingstandard cloning techniques. This construct, known as a “homologousrecombination construct”, can then be introduced into the desired cellseither ex vivo or in vivo.

Gene therapy also can be used to decrease Fhm polypeptide expressionwhere desired by modifying the nucleotide sequence of the endogenouspromoter(s). Such modification is typically accomplished via homologousrecombination methods. For example, a DNA molecule containing all or aportion of the promoter of the Fhm gene(s) selected for inactivation canbe engineered to remove and/or replace pieces of the promoter thatregulate transcription. For example, the TATA box and/or the bindingsite of a transcriptional activator of the promoter may be deleted usingstandard molecular biology techniques; such deletion can inhibitpromoter activity thereby repressing the transcription of thecorresponding Fhm gene. The deletion of the TATA box or thetranscription activator binding site in the promoter may be accomplishedby generating a DNA construct comprising all or the relevant portion ofthe Fhm polypeptide promoter(s) (from the same or a related species asthe Fhm gene(s) to be regulated) in which one or more of the TATA boxand/or transcriptional activator binding site nucleotides are mutatedvia substitution, deletion and/or insertion of one or more nucleotides.As a result, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. The construct, which alsowill typically contain at least about 500 bases of DNA that correspondto the native (endogenous) 5′ and 3′ DNA sequences adjacent to thepromoter segment that has been modified. The construct may be introducedinto the appropriate cells (either ex vivo or in vivo) either directlyor via a viral vector as described herein. Typically, the integration ofthe construct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

Addititional Uses of Fhm Nucleic Acids and Polypeptides

Nucleic acid molecules of the present invention (including those that donot themselves encode biologically active polypeptides) may be used tomap the locations of the Fhm gene and related genes on chromosomes.Mapping may be done by techniques known in the art, such as PCRamplification and in situ hybridization.

Fhm nucleic acid molecules (including those that do not themselvesencode biologically active polypeptides),-may be useful as hybridizationprobes in diagnostic assays to test, either qualitatively orquantitatively, for the presence of an Fhm DNA or corresponding RNA inmammalian tissue or bodily fluid samples.

The Fhm polypeptides may be used (simultaneously or sequentially) incombination with one or more cytokines, growth factors, antibiotics,anti-inflammatories, and/or chemotherapeutic agents as is appropriatefor the indication being treated.

Other methods may also be employed where it is desirable to inhibit theactivity of one or more Fhm polypeptides. Such inhibition may beeffected by nucleic acid molecules which are complementary to and whichhybridize to expression control sequences (triple helix formation) or toFhm mRNA. For example, antisense DNA or RNA molecules, which have asequence that is complementary to at least a portion of the selected Fhmgene(s) can be introduced into the cell. Anti-sense probes may bedesigned by available techniques using the sequence of Fhm polypeptidedisclosed herein. Typically, each such antisense molecule will becomplementary to the start site (5′ end) of each selected Fhm gene. Whenthe antisense molecule then hybridizes to the corresponding Fhm mRNA,translation of this mRNA is prevented or reduced. Anti-sense inhibitorsprovide information relating to the decrease or absence of an Fhmpolypeptide in a cell or organism.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more Fhm polypeptides. In thissituation, the DNA encoding a mutant polypeptide of each selected Fhmpolypeptide can be prepared and introduced into the cells of a patientusing either viral or non-viral methods as described herein. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

In addition, an Fhm polypeptide, whether biologically active or not, maybe used as an immunogen, that is, the polypeptide contains at least oneepitope to which antibodies may be raised. Selective binding agents thatbind to an Fhm polypeptide (as described herein) may be used for in vivoand in vitro diagnostic purposes, including, but not limited to, use inlabeled form to detect the presence of Fhm polypeptide in a body fluidor cell sample. The antibodies may also be used to prevent, treat, ordiagnose a number of diseases and disorders, including those recitedherein. The antibodies may bind to an Fhm polypeptide so as to diminishor block at least one activity characteristic of an Fhm polypeptide, ormay bind to a polypeptide to increase at least one activitycharacteristic of an Fhm polypeptide (including by increasing thepharmacokinetics of the Fhm polypeptide).

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway

EXAMPLE 1 Isolation of DNA Encoding Human Fhm

A TNF family profile search of the Amgen expressed sequence tag (EST)database identified an EST clone designated Fhm 1-00016-g12 from a humanmacrophage cDNA library encoding a potential TNF ligand family member. Afull-length cDNA encoding Fhm was obtained by PCR of first strand cDNAprepared from the 5637 cell line (ATCC #HTB-9) using the followingprimers:

1406-53: 5′ GCCGAGGATCTGGGA CTGA (SEQ ID NO: 1) 1468-66 5′TCGCCAATCCTCCAACCCATCTTA (SEQ ID NO: 2)

The Fhm cDNA comprises 819 nucleotides (SEQ ID NO: 3) and encodes apolypeptide comprising 251 amino acids (SEQ ID NO: 4). Fasta search ofthe SwissProt database with the predicted Fhm protein sequence indicatedthat it is mostly related to TNFa with 28% identity in the C-terminal162 amino acid overlap. Like other TNF ligand family members. Fhm is atype II transmembrane protein, containing a short N-terminalintracellular domain (amino acids 1-36), a hydrophobic transmembraneregion (amino acids 37-56) and a long C-terminal extracellular domain(amino acids 57-251). The C-terminal extracellular domain of Fhmcontained most of the conserved region of the TNF ligand family (Smithet al., Cell 76:952-62, 1994).

EXAMPLE 2 Tissue Specific Expression of Fhm

Tissue specific expression patterns of Fhm gene may be investigated byNorthern blot analysis using a ³²P-labeled PCR product as a probe todetect the presence of Fhm transcript in various tissues.

Cytoplasmic and poly-A+ RNA is isolated from placenta, developingembryos, and various adult tissues using standard techniques Sambrook,J. et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, NewYork (1989). Cells/tissues are lysed with 20 ml of TRIzol reagent (BRL),homogenized for 30 seconds, and extracted with 4 ml of chloroform. Thetubes were centrifuged at 4000 rpm for 30 minutes and the aqueous phasewas transferred to new tubes. RNA was precipitated by adding 10 mlisopropanol, mixing, and centrifuging for 30 minutes at 4200 rpm. TheRNA pellet was washed with 10 ml of 70% ethanol, dried briefly, andresuspended in 0.5 ml TE buffer. Poly A⁺ RNA is prepared by using acommercially available mRNA purification kit (Pharmacia). After elutionof poly A⁺ RNA from the column in 750 μl of TE buffer, the sample wasthen ethanol precipitated by adding 40 μl sample buffer and 1 ml ethanoland maintaining at −70° C. overnight. Poly A⁺ RNA was then fractionatedusing a formaldehyde/agarose gel electrophoresis system. Followingelectrophoresis, the gel is processed and the RNA transferred to a nylonmembrane. See Sambrook et al. Supra. Northern blots are thenprehybridized in 20 ml of prehybridization solution containing 5×SSPE,50% formamide, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denaturedsalmon sperm DNA for 2-4 hours at 42° C. The blots were then hybridizedin 20 ml of hybridization solution containing 6×SSPE, 50% formamide,5×Denhardt's solution, 0.5% SDS, 100 ug/ml denatured salmon sperm DNA.Approximately 5 ng/ml of random primed, ³²P-labeled (RadPrime Kit,GIBCO) Fhm full length cDNA was used as a probe. The blots werehybridized for 18-24 hours at 42° C. The blots were then washed in0.1×SSC, 0.1% SDS at 55° C. The blots were then exposed to x-ray filmsfor three days at 80° C. A weak expression of Fhm was detected in thekidneys.

EXAMPLE 3 Production of Fhm Polypeptides

A. Expression of Fhm Polypeptide in Bacteria

PCR are used to amplify template DNA sequences encoding an Fhmpolypeptide using primers corresponding to the 5′ and 3′ ends of thesequence. The amplified DNA products may be modified to containrestriction enzyme sites to allow for insertion into expression vectors.PCR products are gel purified and inserted into expression vectors usingstandard recombinant DNA methodology. An exemplary vector, such aspAMG21 (ATCC No. 98113) containing the lux promoter and a gene encodingkanamycin resistance is digested with BamHI and NdeI for directionalcloning of inserted DNA. The ligated mixture is transformed into E. colihost strain 393 by electroporation and transformants selected forkanamycin resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of the insert.

Transformed host cells are incubated in 2XYT medium containing 30 μg/mlkanamycin at 30° C. prior to induction. Gene expression can then beinduced by addition of N-(3-oxohexanoyl)-dl-homoserine lactone to afinal concentration of 30 ng/ml followed by incubation at either 30° C.or 37° C. for six hours. Expression of Fhm polypeptide is evaluated bycentrifugation of the culture, resuspension and lysis of the bacterialpellets, and analysis of host cell proteins by SDS-polyacrylamide gelelectrophoresis.

Inclusion bodies containing Fhm polypeptide are purified as follows:Bacterial cells are pelleted by centrifugation and resuspended in water.The cell suspension is lysed by sonication and pelleted bycentrifugation at 195,000×g for 5 to 10 minutes. The supernatant isdiscarded and the pellet washed and transferred to a homogenizer. Thepellet is homogenized in 5 ml. of a Percoll solution (75% liquidPercoll. 0.15M NaCl) until uniformly suspended and then diluted andcentrifuged at 21,600×g for 30 minutes. Gradient fractions containingthe inclusion bodies are recovered and pooled. The isolated inclusionbodies are analyzed by SDS-PAGE. Recombinant Fhm protein was purified aspreviously described (WO 98/46751)

EXAMPLE 4 Production of Anti-Fhm Antibodies

Antibodies to Fhm polypeptides may be obtained by immunization withpurified Fhm protein or with Fhm peptides produced by biological orchemical synthesis. Substantially pure Fhm protein or polypeptide may beisolated from transfected cells as described in Example 3. Concentrationof protein in the final preparation may be adjusted, for example, byconcentration on an amicon filter device, to the level of a fewmicrograms/ml. Monoclonal or polyclonal antibodies to the protein canthen be prepared by any of the procedures known in the art forgenerating antibodies such as those described in Hudson and Bay,“Practical Immunology, Second Edition”, Blackwell ScientificPublications, incorporated herein by reference.

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein described above, which can be unmodified or modifiedto enhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanlarge molecules and may require the use of carriers or adjuvants. Also,host animals vary in response to site of inoculations and dose, withboth inadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng levels) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin.Endocrinol. Metab. 33: 988-991, 1971.

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begin to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology ed. D. Weir, Blackwell, 1973. Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12um). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chapt. 42 in; Manual of Clinical Immunology, 2d Ed. (Rose and Friedman.eds.) Amer. Soc. For Microbiol., Washington, D.C., 1980.

Alternative procedures for obtaining anti-Fhm antibodies may also beemployed, such as immunization of transgenic mice harboring human Igloci for production of fully human antibodies, and screening ofsynthetic antibody libraries, such as those generated by mutagenesis ofan antibody variable domain.

EXAMPLE 5 Functional Analysis of the Role of Fhm

To determine the functional role of Fhm in vivo, the Fhm gene is eitherover expressed in the germ line of animals or inactivated in the germline of mammals by homologous recombination. See, .e.g, U.S. Pat. No.5,489,743 and Interantional Patent Publication No. WO 94/28122,incorporated herein by reference. Animals in which the gene is overexpressed under the regulatory control of exogenous or endogenouspromoter elements are known as transgenic animals. Animals in which anendogenous gene has been inactivated by homologous recombination arealso known as “knockout” animals. Exemplary mammals include rabbits androdent species such as mice.

Transgenic animals allow for the determination of the effect(s) of overexpression or inappropriate expression of the Fhm on development anddisease processes. Fhm transgenic animals can also serve as a modelsystem to test compounds that can modulate receptor mediated Fhmactivity.

The “knockout” animals allow for the determination of the role of Fhm inembryonic development, and in immune and proliferative responses. Therole of Fhm in development, and in immune and proliferative response isdetermined by analysis the effect(s) of gene knockout on the developmentof the embryo as well as on the development and differentiation ofvarious organs and tissues such as the immune system in these animals.(as determined by FACS analysis of cell populations at different stagesof development).

EXAMPLE 6 Specific Recognition of Fhm by Soluble TNF-Receptor FamilyMember NTR3

For receptor binding assay, 2×10⁵ COS-7 cells were seeded in 6-wellplate. The next day, cells were transfected with expression plasmid forFhm by lipofectamin methods according to the manufacturer's instructions(Gibco BRL). The eukaryotic expression vector PCEP4 (Invitrogen) wasused to generate the cDNA contstruct. After 48 hours of transfection,the tissue culture medium was replaced with tissue culture mediumcontaining TNF-receptor family member(s) fused with human IgG Fcportion. After 1 hour incubation at room temperature (RT), cells werewashed three times with 5 ml PBS. Cells were then incubated in DMEMmedium containing 5% BSA and 1:500 dilution of goat anti-human IgG Fcconjugated with alkaline phosphatase (Sigma) for another hour at RT.After three washes with 5 ml TBS buffer, cells were stained with FastRed TR/AS-MX Substrate Kit (Pierce). Positive staining was determined byvisual examination under microscope. Fhm transfected COS-7 cells werespecifically recognized by NTR3Fc fusion protein. The NTR3 protein, amember of the TNF receptor supergene family, is described in detail inco-owned, co-filed provisional U.S. patent application filed Aug. 4,1999, Attorney Docket No. 01017/35549, incorporated herein by referencein its entirety.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

1. An isolated polypeptide comprising the amino acid sequence set forthin SEQ ID NO:
 4. 2. An isolated polypeptide encoded by a nucleic acidselected from the group consisting of: (a) a nucleotide sequence setforth in SEQ ID NO: 3; and (b) a nucleotide sequence encoding thepolypeptide set forth in SEQ ID NO:
 4. 3. An isolated polypeptideproduced by a process of culturing a host cell under suitable conditionsto express the polypeptide and isolating the polypeptide from theculture, wherein the host cell comprises a vector comprising anucleotide sequence that encodes the polypeptide selected from the groupconsisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 3; and(b) a nucleotide sequence encoding the polypeptide set forth in SEQ IDNO:
 4. 4. An isolated polypeptide capable of binding NTR3 and comprisingan amino acid sequence selected from the group consisting of: (a) anamino acid sequence set forth in SEQ ID NO: 4; (b) a fragment of (a)having an N-terminal truncation of up to 100 amino acids; and (c) afragment of (a) or (b) having a C-terminal truncation of up to 75 aminoacids.
 5. An isolated polypeptide comprising an extracellular domainfragment of SEQ ID NO: 4 comprising residues 57 to 251 of SEQ ID NO: 4;wherein the polypeptide is capable of binding to NTR3.
 6. An isolatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, with from 1 to 25 amino acid insertions, wherein the polypeptide iscapable of binding to NTR3.
 7. An isolated polypeptide, wherein theisolated polypeptide binds NTR3, and wherein the isolated polypeptidecomprises an amino acid sequence of SEQ ID NO: 4 with one or more aminoacid substitutions selected from the group consisting of: (a)substitution of the glycine residue at position 145 of SEQ ID NO: 4 withan amino acid selected from the group consisting of proline and alanine;(b) substitution of the tyrosine residue at position 147 of SEQ ID NO: 4with an amino acid selected from the group consisting of tryptophan,phenylalanine, threonine and serine; (c) substitution of the tyrosineresidue at position 150 of SEQ ID NO: 4 with an amino acid selected fromthe group consisting of tryptophan, phenylalanine, threonine and serine;(d) substitution of the serine residue at position 151 of SEQ ID NO: 4with an amino acid selected from the group consisting of threonine,alanine, and cysteine; (e) substitution of the valine residue atposition 153 of SEQ ID NO: 4 with an amino acid selected from the groupconsisting of isoleucine, methionine, leucine, phenylalanine, alanineand norleucine; and (f) substitution of the threonine residue atposition 154 of SEQ ID NO: 4 with an amino acid selected from the groupconsisting of leucine, valine, isoleucine, alanine, and tyrosine.
 8. Apolypeptide comprising a derivative of the polypeptide of any one ofclaims 1 to 7, wherein the polypeptide is capable of binding to NTR3. 9.The polypeptide of any one of claims 1 to 7 that is covalently modifiedwith a water-soluble polymer; wherein the polypeptide is capable ofbinding to NTR3.
 10. The polypeptide of claim 9 wherein thewater-soluble polymer is selected from the group consisting ofpolyethylene glycol, monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyetheylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, and polyvinyl alcohol.
 11. A fusionpolypeptide comprising the polypeptide of any one of claims 1 to 7 fusedto a heterologous amino acid sequence.
 12. The fusion polypeptide ofclaim 11 wherein the heterologous amino acid sequence is an IgG constantdomain or fragment thereof.
 13. A polypeptide of any one of claims 1 to7 further comprising glycosylation; wherein the polypeptide is capableof binding to NTR3.
 14. A composition comprising the polypeptide of anyone of claims 1 to 7, and a pharmaceutically acceptable formulationagent.
 15. The composition of claim 14 wherein the pharmaceuticallyacceptable formulation agent is a carrier, adjuvant, solubilizer,stabilizer, or anti-oxidant.
 16. A method of producing an antibodycomprising using the polypeptide of any one of claims 1 to 7 to generatean antibody capable of binding said polypeptide.
 17. A method ofproducing an antibody comprising using the polypeptide of any one ofclaims 1 to 7 as an immunogen to generate an antibody capable of bindingsaid polypeptide.
 18. A method of producing an antibody comprising usingthe polypeptide of any one of claims 1 to 7 to generate an antibodycapable of binding said polypeptide, the method comprising: (a)injecting a non-human animal with a composition comprising thepolypeptide of any one of claims 1 to 7; and (b) isolating antibodiesfrom the animal, wherein the isolated antibodies includes antibodiesthat specifically bind to the polypeptide.
 19. A method of producing anantibody comprising: (a) immunizing a non-human animal with thepolypeptide of any one of claims 1 to 7; (b) preparing a hybridoma cellline from an antibody-producing cell obtained from the immunized animal,wherein the antibody-producing cell produces antibodies thatspecifically bind the polypeptide; and (c) producing a monoclonalantibody from the hybridoma cell line, wherein the monoclonal antibodyspecifically binds the polypeptide.
 20. A method of selecting anantibody that specifically binds the polypeptide of any one of claims 1to 7, the method comprising: (a) contacting the polypeptide of any oneof claims 1 to 7 with a library, wherein the library comprisesantibodies or fragments thereof; and (b) selecting a member of thelibrary that specifically binds the polypeptide.